Rag-based selection methods for improving aphid resistance in soybeans

ABSTRACT

This invention relates to methods of improving resistance to aphids in soybean plants, as well as methods for identifying and/or selecting soybean plants or germplasm that display improved resistance to one or more biotypes of soybean aphid. In certain examples, the method comprises selecting a first and second soybean plant or germplasm, each of which has a different favorable Rag1, Rag2, or Rag3, allele, haplotype, or marker profile, and crossing those first and second soybean plants to produce a progeny plant with improved soybean aphid resistance. This invention further relates to markers, primers, probes, kits, systems, etc., useful for carrying out the methods described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 13/724154,filed Dec. 21, 2012, now allowed, which claims the benefit of U.S.Provisional Application No. 61/581151, filed Dec. 29, 2011, now expired,the entire content which is herein incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFSWeb as an ASCII formatted sequence listing with a file named“20160202_4377USDIV2_SequenceListing.txt”, created on Feb. 2, 2016, andhaving a size of 43 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of improving resistance to aphids insoybean plants, as well as methods for identifying and/or selectingsoybean plants or germplasm that display improved resistance to one ormore biotypes of soybean aphid.

BACKGROUND

Soybeans (Glycine max L. Merr.) are a major cash crop and investmentcommodity in North America and elsewhere. Soybean oil is one of the mostwidely used edible oils, and soybeans are used worldwide both in animalfeed and in human food production. Additionally, soybean utilization isexpanding to industrial, manufacturing, and pharmaceutical applications.Soybeans are also vulnerable to more than one hundred differentpathogens, with some pathogens having disastrous economic consequences.One important soybean pathogen is the soybean aphid, which can severelyimpact yield. Despite a large amount of effort expended in the art,commercial soybean crops are still largely susceptible to aphidinfestation.

A native of Asia, the soybean aphid (Aphis glycines Matsumura) was firstfound in the Midwest in 2000 (Hartman, G. L., et al., “Occurrence anddistribution of Aphis glycines on soybeans in Illinois in 2000 and itspotential control,” (1 Feb. 2001), available atplantmanagementnetwork.org/phpldefault.asp). It rapidly spreadthroughout the region and into other parts of North America (Patterson,J. and Ragsdale, D., “Assessing and managing risk from soybean aphids inthe North Central States,” (11 Apr. 2002) available atplanthealth.info/aphids_researchupdate.htm). High aphid populations canreduce crop production directly when their feeding causes severe damagesuch as stunting, leaf distortion, and reduced pod set (Sun, Z., et al.,“Study on the uses of aphid-resistant character in wild soybean. I.Aphid-resistance performance of F2 generation from crosses betweencultivated and wild soybeans,” (1990) Soybean Genet. News. 17:43-48).Yield losses attributed to the aphid in some fields in Minnesota during2001, where several thousand aphids occurred on individual soybeanplants, were >50% (Ostlie, K., “Managing soybean aphid,” (2 Oct. 2002),available atsoybeans.umn.edu/crop/insects/aphid/aphid˜publicationmanagingsba.htm),with an average loss of 101 to 202 kg/ha in those fields (Patterson, J.and Ragsdale, D., “Assessing and managing risk from soybean aphids inthe North Central States,” (11 Apr. 2002). In earlier reports fromChina, soybean yields were reduced up to 52% when there was an averageof about 220 aphids per plant (Wang, X.B., et al., “A study on thedamage and economic threshold of the soybean aphid at the seedlingstage,” (1994) Plant Prot. (China) 20:12-13), and plant height wasdecreased by about 210 mm after severe aphid infestation (Wang, X. B.,et al., “Study on the effects of the population dynamics of soybeanaphid (Aphis glycines) on both growth and yield of soybean,” (1996)Soybean Sci. 15:243-247). An additional threat posed by the aphid is itsability to transmit certain plant viruses to soybean, such as Alfalfamosaic virus, Soybean dwarf virus, and Soybean mosaic virus (Sama, S.,et al., “Varietal screening for resistance to the aphid, Aphis glycines,in soybean,” (1974) Research Reports 1968-1974, pp. 171-172; Iwaki, M.,et al., “A persistent aphid borne virus of soybean, Indonesian Soybeandwarf virus transmitted by Aphis glycines,” (1980) Plant Dis.64:1027-1030; Hartman, G.L., et al., “Occurrence and distribution ofAphis glycines on soybeans in Illinois in 2000 and its potentialcontrol,” (1 Feb. 2001), available atplantmanagementnetwork.org/phpldefault.asp; Hill, J. H., et al., “Firstreport of transmission of Soybean mosaic virus and Alfalfa mosaic virusby Aphis glycines (Homoptera, Aphididae),” (1996) Appl. Entomol. 2001.31:178-180; Clark, A. J. and Perry, K. L., “Transmissibility of fieldisolates of soybean viruses by Aphis glycines,” (2002) Plant Dis.86:1219-1222).

Currently, millions of dollars are spent annually on sprayinginsecticides to control soybean aphid infestation. An integral componentof an integrated pest management (IPM) program to control aphids isplant resistance (Auclair, J. L., “Host plant resistance,” pp. 225-265In P. Harrewijn (ed.) Aphids: Their biology, natural enemies, andcontrol, Vol. C., Elsevier, New York (1989); Harrewijn, P. and Minks, A.K., “Integrated aphid management: General aspects,” pp. 267-272, In A.K. Minks and P. Harrewijn (ed.) Aphids: Their biology, natural enemies,and control, Vol. C., Elsevier, New York (1989)). Insect resistance cansignificantly reduce input costs for producers (Luginbill, J. P.,“Developing resistant plants—The ideal method of controlling insects,”(1969) USDA, ARS. Prod. Res. Rep. 111, USGPO, D.C.).

There are currently three well-documented biotypes (i.e., a subspeciesof soybean aphid that shares certain genetic traits or a specifiedgenotype) of soybean aphid that have been collected in Urbana, Ill.(biotype 1), Wooster, Ohio (biotype 2), and Indiana (biotype 3).Additionally, there are three kinds of plant resistance that have beenidentified: antibiosis, antixenosis, and tolerance. Antibiosis(non-choice) is the plant's ability to reduce the survival,reproduction, and fecundity of the insect. Antixenosis (choice) is theplant's ability to deter the insect from feeding or identifying theplant as a food source. Tolerance is the plant's ability to withstandheavy infestation without significant yield loss.

To date, three different soybean aphid resistance genes have beenidentified and mapped to the soybean genome. Rag1 was the first soybeanresistance gene identified (Mian, et al., Genetic linkage mapping of thesoybean aphid resistance gene in PI 243540, Theor. Appl. Genet.117:955-962 (2008)). Rag1 has been mapped to linkage group M in thevicinity of SSR markers Satt540 and Satt463 (Kim, et al., Fine mappingof the soybean aphid resistance gene Rag1 in soybean, Theor. Appl.Genet., 120:1063-1071 (2010)). Rag2 has been mapped to linkage group Fin the vicinity of SSR markers Satt334 and Sct_033 (Mian, et al.,Genetic linkage mapping of the soybean aphid resistance gene in PI243540, Theor. Appl. Genet. 117:955-962 (2008)). Rag3 is located onlinkage group J in the vicinity of markers Sat_339 and Sat_370. It hasalso been previously determined that some aphid biotypes are resistantto certain of the Rag genes but are susceptible to others (Mian, et al.,Genetic linkage mapping of the soybean aphid resistance gene in PI243540, Theor. Appl. Genet. 117:955-962 (2008)).

Molecular markers have been used to selectively improve soybean cropsthrough the use of marker assisted selection. Any detectible polymorphictrait can be used as a marker so long as it is inherited differentiallyand exhibits linkage disequilibrium with a phenotypic trait of interest.A number of soybean markers have been mapped and linkage groups created,as described in Cregan, P. B., et al., “An Integrated Genetic LinkageMap of the Soybean Genome” (1999) Crop Science 39:1464-90, and morerecently in Choi, et al., “A Soybean Transcript Map: Gene Distribution,Haplotype and Single-Nucleotide Polymorphism Analysis” (2007) Genetics176:685-96. Many soybean markers are publicly available at the USDAaffiliated soybase website (www.soybase.org).

Most plant traits of agronomic importance are polygenic, otherwise knownas quantitative, traits. A quantitative trait is controlled by severalgenes located at various locations, or loci, in the plant's genome. Themultiple genes have a cumulative effect which contributes to thecontinuous range of phenotypes observed in many plant traits. Thesegenes are referred to as quantitative trait loci (QTL). Recombinationfrequency measures the extent to which a molecular marker is linked witha QTL. Lower recombination frequencies, typically measured incentiMorgans (cM), indicate greater linkage between the QTL and themolecular marker. The extent to which two features are linked is oftenreferred to as the genetic distance. The genetic distance is alsotypically related to the physical distance between the marker and theQTL; however, certain biological phenomenon (including recombinational“hot spots”) can affect the relationship between physical distance andgenetic distance. Generally, the usefulness of a molecular marker isdetermined by the genetic and physical distance between the marker andthe selectable trait of interest.

In some cases, multiple closely linked markers, such as SingleNucleotide Polymorphism (SNP) markers, can be found to exist in acertain region of a plant genome encompassing one or more QTL. In suchcases, by determining the allele present at each of those marker loci, ahaplotype for that region of the plant genome can be determined.Further, by determining alleles or haplotypes present at multipleregions of the plant genome related to the same phenotypic trait, amarker profile for that trait can be determined. Such haplotype andmarker profile information can be useful in identifying and selectingplants with certain desired traits.

There remains a need for soybean plants with improved resistance tosoybean aphid and methods for identifying and selecting such plants.

SUMMARY

This invention relates to methods of improving resistance to aphids insoybean plants, as well as methods for identifying and/or selectingsoybean plants or germplasm that display improved resistance to one ormore biotypes of soybean aphid. In certain examples, the methodcomprises selecting a first and second soybean plant or germplasm, eachof which has a different favorable Rag1, Rag2, or Rag3, allele,haplotype, or marker profile, and crossing those first and secondsoybean plants to produce a progeny plant with stacked Rag alleles,haplotypes, or marker profiles and improved soybean aphid resistance. Inother examples, the method comprises detecting a stacked marker profilecomprising two or more Rag haplotypes. This invention further relates tomarkers, primers, probes, kits, systems, etc., useful for carrying outthe methods described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate a partial genetic map of soybean illustrating therelative map position of the Rag intervals and numerous linked markerloci. FIG. 1A illustrates a genetic map of linkage group M and therelative map position of the Rag1 interval. FIG. 1B illustrates agenetic map of linkage group F and the relative map position of the Rag2interval. FIG. 1C illustrates a genetic map of linkage group J and therelative map position of the Rag3 interval.

SUMMARY OF THE SEQUENCES

SEQ ID NOs: 1-4 comprise nucleotide sequences of regions of the soybeangenome, each capable of being used as a probe or primer, either alone orin combination, for the detection of marker locus S14181-1-Q1 on LG-M.In certain examples, SEQ ID NOs: 1 and 2 are used as primers while SEQID NOs: 3 and 4 are used as probes.

SEQ ID NO: 5 is the genomic DNA region encompassing marker locusS14181-1-Q1 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 6-9 comprise nucleotide sequences of regions of the soybeangenome, each capable of being used as a probe or primer, either alone orin combination, for the detection of marker locus S13871-1-Q1 on LG-M.In certain examples, SEQ ID NOs: 6 and 7 are used as primers while SEQID NOs: 8 and 9 are used as probes.

SEQ ID NO: 10 is the genomic DNA region encompassing marker locusS13871-1-Q1 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 11-14 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S14161-1-Q10on LG-M. In certain examples, SEQ ID NOs: 11 and 12 are used as primerswhile SEQ ID NOs: 13 and 14 are used as probes.

SEQ ID NO: 15 is the genomic DNA region encompassing marker locusS14161-1-Q10 on LG-M. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 16-19 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S09515-1-Q1on LG-M. In certain examples, SEQ ID NOs: 16 and 17 are used as primerswhile SEQ ID NOs: 18 and 19 are used as probes.

SEQ ID NO: 20 is the genomic DNA region encompassing marker locusS09515-1-Q1 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 21-24 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S14151-1-Q1on LG-M. In certain examples, SEQ ID NOs: 21 and 22 are used as primerswhile SEQ ID NOs: 23 and 24 are used as probes.

SEQ ID NO: 25 is the genomic DNA region encompassing marker locusS14151-1-Q1 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 26-29 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S14151-2-Q4on LG-M. In certain examples, SEQ ID NOs: 26 and 27 are used as primerswhile SEQ ID NOs: 28 and 29 are used as probes.

SEQ ID NO: 30 is the genomic DNA region encompassing marker locusS14151-2-Q4 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 31-34 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S07164-1-Q12on LG-M. In certain examples, SEQ ID NOs: 31 and 32 are used as primerswhile SEQ ID NOs: 33 and 34 are used as probes.

SEQ ID NO: 35 is the genomic DNA region encompassing marker locusS07164-1-Q12 on LG-M. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 36-39 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S14182-1-Q1on LG-M. In certain examples, SEQ ID NOs: 36 and 37 are used as primerswhile SEQ ID NOs: 38 and 39 are used as probes.

SEQ ID NO: 40 is the genomic DNA region encompassing marker locusS14182-1-Q1 on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 41-44 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S00812-1-A onLG-M. In certain examples, SEQ ID NOs: 41 and 42 are used as primerswhile SEQ ID NOs: 43 and 44 are used as probes.

SEQ ID NO: 45 is the genomic DNA region encompassing marker locusS00812-1-A on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 46-49 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S02780-1-A onLG-M. In certain examples, SEQ ID NOs: 46 and 47 are used as primerswhile SEQ ID NOs: 48 and 49 are used as probes.

SEQ ID NO: 50 is the genomic DNA region encompassing marker locusS02780-1-A on LG-M. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 51-54 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS14761-001-Q001 on LG-F. In certain examples, SEQ ID NOs: 51 and 52 areused as primers while SEQ ID NOs: 53 and 54 are used as probes.

SEQ ID NO: 55 is the genomic DNA region encompassing marker locusS14761-001-Q001 on LG-F. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 56-59 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS14771-001-Q001 on LG-F. In certain examples, SEQ ID NOs: 56 and 57 areused as primers while SEQ ID NOs: 58 and 59 are used as probes.

SEQ ID NO: 60 is the genomic DNA region encompassing marker locus S14771-001-Q001 on LG-F. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 61-64 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S07165-1-Q3on LG-F. In certain examples, SEQ ID NOs: 61 and 62 are used as primerswhile SEQ ID NOs: 63 and 64 are used as probes.

SEQ ID NO: 65 is the genomic DNA region encompassing marker locusS07165-1-Q3 on LG-F. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 66-69 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS14778-001-Q001 on LG-F. In certain examples, SEQ ID NOs: 66 and 67 areused as primers while SEQ ID NOs: 68 and 69 are used as probes.

SEQ ID NO: 70 is the genomic DNA region encompassing marker locusS14778-001-Q001 on LG-F. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 71-74 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S01164-1-Q1on LG-F. In certain examples, SEQ ID NOs: 71 and 72 are used as primerswhile SEQ ID NOs: 73 and 74 are used as probes.

SEQ ID NO: 75 is the genomic DNA region encompassing marker locusS01164-1-Q1 on LG-F. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 76-83 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS13662-1-Q3/Q6 on LG-J. In certain examples, SEQ ID NOs: 76 and 77 areused as primers while SEQ ID NOs: 78 and 79 are used as probes toamplify and detect S13662-1-Q3. In other examples, SEQ ID NOs: 80 and 81are used as primers while SEQ ID NOs: 82 and 83 are used as probes toamplify and detect S13662-1-Q6.

SEQ ID NO: 84 is the genomic DNA region encompassing marker locusS13662-1-Q3/Q6 on LG-J. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 85-88 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S13663-1-Q1on LG-J. In certain examples, SEQ ID NOs: 85 and 86 are used as primerswhile SEQ ID NOs: 87 and 88 are used as probes.

SEQ ID NO: 89 is the genomic DNA region encompassing marker locusS13663-1-Q1 on LG-J. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 90-93 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S11411-1-Q1on LG-J. In certain examples, SEQ ID NOs: 90 and 91 are used as primerswhile SEQ ID NOs: 92 and 93 are used as probes.

SEQ ID NO: 94 is the genomic DNA region encompassing marker locusS11411-1-Q1 on LG-J. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 95-102 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS13664-1-Q1/Q002 on LG-J. In certain examples, SEQ ID NOs: 95 and 96 areused as primers while SEQ ID NOs: 97 and 98 are used as probes toamplify and detect S13664-1-Q1. In other examples, SEQ ID NOs: 99 and100 are used as primers while SEQ ID NOs: 101 and 102 are used as probesto amplify and detect S13664-1-Q002.

SEQ ID NO: 103 is the genomic DNA region encompassing marker locusS13664-1-Q002on LG-J. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 104-113 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS13672-1-Q1/Q2/Q3 on LG-J. In certain examples, SEQ ID NOs: 104 and 105are used as primers while SEQ ID NOs: 106 and 107 are used as probes toamplify and detect S13672-1-Q1. In other examples, SEQ ID NOs: 108 and109 are used as primers while SEQ ID NOs: 106 and 107 are used as probesto amplify and detect S13672-1-Q2. In still further examples, SEQ IDNOs: 110 and 111 are used as primers while SEQ ID NOs: 112 and 113 areused as probes to amplify and detect S13672-1-Q3.

SEQ ID NO: 114 is the genomic DNA region encompassing marker locusS13672-1-Q1/Q2/Q3 on LG-J. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 115-120 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS13674-1-Q1/Q007 on LG-J. In certain examples, SEQ ID NOs: 115 and 116are used as primers while SEQ ID NOs: 117 and 118 are used as probes toamplify and detect S13674-1-Q1. In other examples, SEQ ID NOs: 119 and120 are used as primers while SEQ ID NOs: 117 and 118 are used as probesto amplify and detect S13674-1-Q007.

SEQ ID NO: 121 is the genomic DNA region encompassing marker locusS13674-1-Q1/Q007 on LG-J. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 122-125 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S13675-2-Q1on LG-J. In certain examples, SEQ ID NOs: 122 and 123 are used asprimers while SEQ ID NOs: 124 and 125 are used as probes.

SEQ ID NO: 126 is the genomic DNA region encompassing marker locus S13675-2-Q1 on LG-J. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 127-130 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locus S01190-1-A onLG-F. In certain examples, SEQ ID NOs: 127 and 128 are used as primerswhile SEQ ID NOs: 129 and 130 are used as probes.

SEQ ID NO: 131 is the genomic DNA region encompassing marker locusS01190-1-A on LG-F. In certain examples this sequence is used to designprimers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

SEQ ID NOs: 132-135 comprise nucleotide sequences of regions of thesoybean genome, each capable of being used as a probe or primer, eitheralone or in combination, for the detection of marker locusS25354-001-Q001 on LG-M. In certain examples, SEQ ID NOs: 132 and 133are used as primers while SEQ ID NOs: 134 and 135 are used as probes.

SEQ ID NO: 136 is the genomic DNA region encompassing marker locusS15354-001-Q001 on LG-M. In certain examples this sequence is used todesign primers and probes directed toward this marker. In certain otherexamples this sequence, or a portion of it, is used as a probe to detectthis marker.

DETAILED DESCRIPTION

In one embodiment, a novel method is provided for improving resistanceto one or more soybean aphid biotypes in a soybean germplasm, plant,line, or strain, the method comprising a) selecting a first soybeanplant or germplasm, or a progeny thereof, wherein said first soybeanplant, germplasm, or progeny comprises a favorable allele, haplotype, ormarker profile of at least one of Rag1, Rag2, and Rag3; and selecting asecond soybean plant or germplasm, or a progeny thereof, wherein saidsecond soybean plant, germplasm, or progeny comprises a favorableallele, haplotype, or marker profile of at least one of Rag1, Rag2, andRag3 different than that of said first soybean plant, germplasm, orprogeny; and b) crossing the first soybean plant, germplasm, or progenywith the second soybean plant, germplasm, or progeny to produce aprogeny plant with stacked Rag alleles, haplotypes, or marker profilesand improved soybean aphid resistance.

In some examples, the Rag1 haplotype comprises one or more markers thatfall within the interval flanked by and including Satt435 and Sat_244,the Rag2 haplotype comprises one or more markers that fall within theinterval flanked by and including Satt334 and Satt510, and/or the Rag3haplotype comprises one or more markers that fall within the intervalflanked by and including Sat_339 and Sat_370. In other examples, theRag1 haplotype comprises one or more markers that fall within theinterval flanked by and including physical position S5464314-8194502 onLG-M on the Glymal soybean genome assembly (see, e.g., Schmutz J, et al.(2010). “Genome sequence of the palaeopolyploid soybean.” Nature 463,178-183; see also, www.phytozome.net/soybean; each of which isincorporated herein by reference in its entirety), the Rag2 haplotypecomprises one or more markers that fall within the interval flanked byand including physical position 28416122-30590233 on LG-F on the Glymalsoybean genome assembly, and/or the Rag3 haplotype comprises one or moremarkers that fall within the interval flanked by and including physicalposition 4157916-7054678 on LG-J on the Glymal soybean genome assembly.

In further examples, the at least one Rag haplotype comprises markerloci selected from the group consisting of: (a) one or more marker lociselected from the group consisting of S15354-001-Q001, S14181-1-Q1,S13871-1-Q1, S14161-1-Q10, S09515-1-Q1, S14151-1-Q1, S14151-2-Q4,S07164-1-Q12, S14182-1-Q1, S00812-1-A, and S02780-1-A; (b) one or moremarker loci selected from the group consisting of S01190-1-A,S14761-001-Q001, S14771-001-Q001, S07165-1-Q3, S14778-001-Q001, andS01164-1-Q1; and (c) one or more marker loci selected from the groupconsisting of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.In still further examples, the at least one Rag haplotype comprises twoor more of the marker loci within one or more of (a), (b), or (c). Inother examples, the at least one Rag haplotype comprises three or moreof the marker loci within one or more of (a), (b), or (c). In yet otherexamples, the at least one Rag haplotype comprises four or more of themarker loci within one or more of (a), (b), or (c). In even furtherexamples, the at least one Rag haplotype comprises all of the markerloci within one or more of (a), (b), or (c).

In certain examples the first soybean plant, germplasm, or progeny andsaid second soybean plant, germplasm, or progeny collectively comprise afavorable allele, haplotype, or marker profile of all three of Rag1,Rag2, and Rag3.

In other examples, the improved soybean aphid resistance compriseseither improved antibiosis resistance or improved antixenosisresistance. In yet other examples, the improved soybean aphid resistancecomprises both improved antibiosis resistance and improved antixenosisresistance. In additional examples, the improved soybean aphidresistance comprises improved resistance to at least two soybean aphidbiotypes, at least three soybean aphid phenotypes, at least four soybeanaphid phenotypes, or at least five soybean aphid biotypes.

In additional examples, the method further comprises detecting thefavorable Rag allele, the detecting step comprising amplifying a markernucleic acid or a portion of the marker nucleic acid. In other examples,the method further comprises detecting the resulting amplified markernucleic acid. In certain particular examples, the amplifying comprisesperforming a polymerase chain reaction (PCR) using one or more nucleicacids from the plant as a template in the PCR.

In some particular examples, the method comprises amplifying at least aportion of one or more genome regions selected from the group consistingof SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 84, 89, 94, 103, 114, 121, 126, 131, and 136. In other examples, theprimer or primer pair comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21, 22,26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51, 52, 56, 57, 61, 62, 66, 67,71, 72, 76, 77, 80, 81, 85, 86, 90, 91, 95, 96, 99, 100, 104, 105, 108,109, 110, 111, 115, 116, 119, 120, 122, 123, 127, 128, 132, and 133.

In certain other examples, the detecting further comprises providing adetectable probe. In some particular examples, the probe comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:3, 4, 8, 9, 13, 14, 18, 19, 23, 24, 28, 29, 33, 34, 38, 39, 43, 44, 48,49, 53, 54, 58, 59, 63, 64, 68, 69, 73, 74, 78, 79, 82, 83, 87, 88, 92,93, 97, 98, 101, 102, 106, 107, 112, 113, 117, 118, 124, 125, 129, 130,134, and 135. In other examples, the probe comprises at least a portionof a nucleic acid sequence selected from the group consisting of SEQ IDNOs: 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 84, 89,94, 103, 114, 121, 126, 131, and 136.

In further embodiments, the invention relates to a method of selecting asoybean plant or germplasm that displays improved resistance to one ormore soybean aphid biotypes. In certain examples, the improvedresistance comprises one or more of improved antibiosis resistance orimproved antixenosis resistance. In other examples, the improvedresistance comprises both improved antibiosis and antixenosisresistance. In further examples, the improved soybean aphid resistancecomprises improved resistance to at least two soybean aphid biotypes. Inother examples, the improved soybean aphid resistance comprises improvedresistance to at least three soybean aphid biotypes. In still furtherexamples, the improved soybean aphid resistance comprises improvedresistance to at least four soybean aphid biotypes or at least fivesoybean aphid biotypes.

In certain examples, the method comprises detecting in a first soybeanplant or germplasm, or a part thereof, a favorable allele of at leasttwo of Rag1, Rag2, and Rag3; and selecting the first soybean plant orgermplasm, or selecting a progeny of the first soybean plant orgermplasm. In additional examples, the detecting involves detection of afavorable allele of all three of Rag1, Rag2, and Rag3.

In certain particular examples, the detecting comprises amplifying amarker nucleic acid or a portion of the marker nucleic acid anddetecting the resulting amplified marker nucleic acid. In additionalexamples, the amplifying comprises performing a polymerase chainreaction (PCR) using one or more nucleic acid from the plant as atemplate in the PCR.

In yet further examples, the method further comprises crossing theselected first soybean plant or germplasm with a second soybean plant orgermplasm. In particular examples, the second soybean plant or germplasmcomprises an exotic soybean strain or an elite soybean strain.

Plants, including soybean plants, seeds, tissue cultures, variants andmutants, having improved soybean aphid resistance are also provided. Incertain examples, plants produced by the foregoing methods are provided.In other examples, elite lines having a stacked Rag haplotype areprovided. In additional examples, plants comprising a stack comprisingone or more the Rag haplotypes or marker profiles discussed herein areprovided. In yet further examples, plants comprising a stack comprisingone or more favorable or disfavored alleles at the marker loci discussedherein are provided. In certain examples, plants comprising stackcomprising one or more Rag haplotype selected from the group consistingof Rag1-b, Rag1-c, Rag2-d, Rag3-b, and Rag3-d are provided. In certainother examples, plants comprising a stack comprising a haplotype ormarker profile selected from the group consisting of (a) Rag1-b/Rag3-b;(b) Rag1-b; (c) Rag1-c/Rag3-d; (d) Rag1-e; and (e) Rag1-d/Rag2-c areprovided. In some specific examples, plants are provided that have aRag1/Rag-2 stack and that possess improved resistance to aphid biotype3. In yet further examples, plants comprising a stack comprising one ormore favorable or disfavored allele at (a) one or more marker lociselected from the group consisting S15354-001-Q001, S14181-1-Q1,S13871-1-Q1, S14161-1-Q10, S09515-1-Q1, S14151-1-Q1, S14151-2-Q4,S07164-1-Q12, S14182-1-Q1, S00812-1-A, and S02780-1-A; (b) one or moremarker loci selected from the group consisting of S01190-1-A,S14761-001-Q001, S14771-001-Q001, S07165-1-Q3, S14778-001-Q001, andS01164-1-Q1; or (c) one or more marker loci selected from the groupconsisting of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1are provided. In further examples, plants comprising a stack comprisingone or more favorable or disfavored allele at (a) the marker lociS14161-1-Q10, S09515-1-Q1, S14151-2-Q4, and S07164-1-Q12; (b) the markerloci S07165-1-Q3, S01190-1-A, and S01164-1-Q1; or (c) the marker lociS11411-1-Q1, S13674-1-Q1/Q007, and S13675-2-Q1 are provided.

Also provided are isolated nucleic acids, kits, and systems useful forthe identification and selection methods disclosed herein. In certainexamples, isolated nucleic acids, kits, and systems useful for thedetection of the Rag haplotypes or marker profiles discussed herein areprovided. In yet further examples, isolated nucleic acids, kits, andsystems useful for the detection of the favorable or disfavored allelesat the marker loci discussed herein are provided. In certain examples,isolated nucleic acids, kits, and systems useful for the detection of aRag haplotype selected from the group consisting of Rag1-b, Rag1-c,Rag2-d, Rag3-b, and Rag3-d are provided. In certain other examples,isolated nucleic acids, kits, and systems useful for the detection of ahaplotype or marker profile selected from the group consisting of (a)Rag1-b/Rag3-b; (b) Rag1-b; (c) Rag1-c/Rag3-d; (d) Rag1-e; and (e)Rag1-d/Rag2-c are provided. In yet further examples, isolated nucleicacids, kits, and systems useful for the detection of a favorable ordisfavored allele at (a) one or more marker loci selected from the groupconsisting S15354-001-Q001, S14181-1-Q1, S13871-1-Q1, S14161-1-Q10,S09515-1-Q1, S14151-1-Q1, S14151-2-Q4, S07164-1-Q12, S14182-1-Q1,S00812-1-A, and S02780-1-A; (b) one or more marker loci selected fromthe group consisting of S01190-1-A, S14761-001-Q001, S14771-001-Q001,S07165-1-Q3, S14778-001-Q001, and S01164-1-Q1; or (c) one or more markerloci selected from the group consisting of S13662-1-Q3/Q6, S13663-1-Q1,S11411-1-Q1, S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, andS13675-2-Q1 are provided. In further examples, isolated nucleic acids,kits, and systems useful for the detection of a favorable or disfavoredallele at (a) the marker loci S14161-1-Q10, S09515-1-Q1, S14151-2-Q4,and S07164-1-Q12; (b) the marker loci S07165-1-Q3, S01190-1-A, andS01164-1-Q1; or (c) the marker loci S11411-1-Q1, S13674-1-Q1/Q007, andS13675-2-Q1 are provided.

It is to be understood that this invention is not limited to particularembodiments, which can, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.Further, all publications referred to herein are incorporated byreference herein for the purpose cited to the same extent as if each wasspecifically and individually indicated to be incorporated by referenceherein.

Definitions:

As used in this specification and the appended claims, terms in thesingular and the singular forms “a,” “an,” and “the,” for example,include plural referents unless the content clearly dictates otherwise.Thus, for example, reference to “plant,” “the plant,” or “a plant” alsoincludes a plurality of plants; also, depending on the context, use ofthe term “plant” can also include genetically similar or identicalprogeny of that plant; use of the term “a nucleic acid” optionallyincludes, as a practical matter, many copies of that nucleic acidmolecule; similarly, the term “probe” optionally (and typically)encompasses many similar or identical probe molecules.

Additionally, as used herein, “comprising” is to be interpreted asspecifying the presence of the stated features, integers, steps, orcomponents as referred to, but does not preclude the presence oraddition of one or more features, integers, steps, or components, orgroups thereof. Thus, for example, a kit comprising one pair ofoligonucleotide primers may have two or more pairs of oligonucleotideprimers. Additionally, the term “comprising” is intended to includeexamples encompassed by the terms “consisting essentially of” and“consisting of.” Similarly, the term “consisting essentially of” isintended to include examples encompassed by the term “consisting of.”

Certain definitions used in the specification and claims are providedbelow. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

“Agronomics,” “agronomic traits,” and “agronomic performance” refer tothe traits (and underlying genetic elements) of a given plant varietythat contribute to yield over the course of a growing season. Individualagronomic traits include emergence vigor, vegetative vigor, stresstolerance, disease resistance or tolerance, insect resistance ortolerance, herbicide resistance, branching, flowering, seed set, seedsize, seed density, standability, threshability, and the like.

“Allele” means any of one or more alternative forms of a geneticsequence. In a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes. With regard to a SNP marker, allele refers to the specificnucleotide base present at that SNP locus in that individual plant.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods, such as theligase chain reaction (LCR), and RNA polymerase based amplification(e.g., by transcription) methods. An “amplicon” is an amplified nucleicacid, e.g., a nucleic acid that is produced by amplifying a templatenucleic acid by any available amplification method (e.g., PCR, LCR,transcription, or the like).

An “ancestral line” is a parent line used as a source of genes, e.g.,for the development of elite lines.

An “ancestral population” is a group of ancestors that have contributedthe bulk of the genetic variation that was used to develop elite lines.

“Backcrossing” is a process in which a breeder crosses a progeny varietyback to one of the parental genotypes one or more times.

“Biotype” or “aphid biotype” means a subspecies of soybean aphid thatshare certain genetic traits or a specified genotype. There arecurrently three well-documented biotypes of soybean aphid: Urbana, Ill.(biotype 1), Wooster, Ohio (biotype 2), and Indiana (biotype 3). Anadditional biotype, referred to herein as biotype X, was collected fromsoybean fields in Lime Springs, Iowa. A further biotype, referred toherein as biotype Y, was collected from a field near Lomira, Wis. insummer 2011.

The term “chromosome segment” designates a contiguous linear span ofgenomic DNA that resides in planta on a single chromosome.

“Cultivar” and “variety” are used synonymously and mean a group ofplants within a species (e.g., Glycine max) that share certain genetictraits that separate them from other possible varieties within thatspecies. Soybean cultivars are inbred lines produced after severalgenerations of self-pollinations. Individuals within a soybean cultivarare homogeneous, nearly genetically identical, with most loci in thehomozygous state.

An “elite line” is an agronomically superior line that has resulted frommany cycles of breeding and selection for superior agronomicperformance. Numerous elite lines are available and known to those ofskill in the art of soybean breeding.

An “elite population” is an assortment of elite individuals or linesthat can be used to represent the state of the art in terms ofagronomically superior genotypes of a given crop species, such assoybean.

An “exotic soybean strain” or an “exotic soybean germplasm” is a strainor germplasm derived from a soybean not belonging to an available elitesoybean line or strain of germplasm. In the context of a cross betweentwo soybean plants or strains of germplasm, an exotic germplasm is notclosely related by descent to the elite germplasm with which it iscrossed. Most commonly, the exotic germplasm is not derived from anyknown elite line of soybean, but rather is selected to introduce novelgenetic elements (typically novel alleles) into a breeding program.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form.

“Genotype” refers to the genetic constitution of a cell or organism.

“Germplasm” means the genetic material that comprises the physicalfoundation of the hereditary qualities of an organism. As used herein,germplasm includes seeds and living tissue from which new plants may begrown; or, another plant part, such as leaf, stem, pollen, or cells,that may be cultured into a whole plant. Germplasm resources providesources of genetic traits used by plant breeders to improve commercialcultivars.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes). Anindividual is “heterozygous” if more than one allele type is present ata given locus (e.g., a diploid individual with one copy each of twodifferent alleles). The term “homogeneity” indicates that members of agroup have the same genotype at one or more specific loci. In contrast,the term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

“Introgression” means the entry or introduction of a gene, QTL,haplotype, marker profile, trait, or trait locus from the genome of oneplant into the genome of another plant.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor. Traditionally, a subline has been derived by inbreeding theseed from an individual soybean plant selected at the F3 to F5generation until the residual segregating loci are “fixed” or homozygousacross most or all loci. Commercial soybean varieties (or lines) aretypically produced by aggregating (“bulking”) the self-pollinatedprogeny of a single F3 to F5 plant from a controlled cross between 2genetically different parents. While the variety typically appearsuniform, the self-pollinating variety derived from the selected planteventually (e.g., F8) becomes a mixture of homozygous plants that canvary in genotype at any locus that was heterozygous in the originallyselected F3 to F5 plant. Marker-based sublines that differ from eachother based on qualitative polymorphism at the DNA level at one or morespecific marker loci are derived by genotyping a sample of seed derivedfrom individual self-pollinated progeny derived from a selected F3-F5plant. The seed sample can be genotyped directly as seed, or as planttissue grown from such a seed sample. Optionally, seed sharing a commongenotype at the specified locus (or loci) are bulked providing a sublinethat is genetically homogenous at identified loci important for a traitof interest (e.g., yield, tolerance, etc.).

“Linkage” refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent. Genetic recombination occurs with anassumed random frequency over the entire genome. Genetic maps areconstructed by measuring the frequency of recombination between pairs oftraits or markers. The closer the traits or markers are to each other onthe chromosome, the lower the frequency of recombination, and thegreater the degree of linkage. Traits or markers are considered hereinto be linked if they generally co-segregate. A 1/100 probability ofrecombination per generation is defined as a map distance of 1.0centiMorgan (1.0 cM).

The genetic elements or genes located on a single chromosome segment arephysically linked. Advantageously, the two loci are located in closeproximity such that recombination between homologous chromosome pairsdoes not occur between the two loci during meiosis with high frequency,e.g., such that linked loci co-segregate at least about 90% of the time,e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, ormore of the time. The genetic elements located within a chromosomesegment are also genetically linked, typically within a geneticrecombination distance of less than or equal to 50 centimorgans (cM),e.g., about 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM orless. That is, two genetic elements within a single chromosome segmentundergo recombination during meiosis with each other at a frequency ofless than or equal to about 50%, e.g., about 49%, 40%, 30%, 20%, 10%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25% or less. Closely linkedmarkers display a cross over frequency with a given marker of about 10%or less (the given marker is within about 10 cM of a closely linkedmarker). Put another way, closely linked loci co-segregate at leastabout 90% of the time.

When referring to the relationship between two genetic elements, such asa genetic element contributing to resistance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the resistance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for resistance) is physically linked with an “unfavorable” allele atthe proximal marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

“Linkage disequilibrium” refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

“Linkage group” refers to traits or markers that generally co-segregate.A linkage group generally corresponds to a chromosomal region containinggenetic material that encodes the traits or markers. As such, a linkagegroup can generally be assigned to a certain chromosome, and suchassociations are well known in the art, for example from the soybasedatabase (soybase.org). For example, soybean LG-M corresponds to soybeanchromosome 7, soybean LG-F corresponds to soybean chromosome 13, andsoybean LG-J corresponds to soybean chromosome 16.

“Locus” is a defined segment of DNA.

A “map location” is an assigned location on a genetic map relative tolinked genetic markers where a specified marker can be found within agiven species.

“Mapping” is the process of defining the linkage relationships of locithrough the use of genetic markers, populations segregating for themarkers, and standard genetic principles of recombination frequency.

“Marker” or “molecular marker” is a term used to denote a nucleic acidor amino acid sequence that is sufficiently unique to characterize aspecific locus on the genome. Examples include Restriction FragmentLength Polymorphisms (RFLPs), Single Sequence Repeats (SSRs), TargetRegion Amplification Polymorphisms (TRAPs), Isozyme Electrophoresis,Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Single NucleotidePolymorphisms (SNPs). Additionally, other types of molecular markers areknown in the art, and phenotypic traits may also be used as markers inthe methods. All markers are used to define a specific locus on thesoybean genome. Large numbers of these markers have been mapped (see,e.g., the Soybase database at soybase.org). Each marker is therefore anindicator of a specific segment of DNA, having a unique nucleotidesequence. The map positions provide a measure of the relative positionsof particular markers with respect to one another. When a trait isstated to be linked to a given marker it will be understood that theactual DNA segment whose sequence affects the trait generallyco-segregates with the marker. More precise and definite localization ofa trait can be obtained if markers are identified on both sides of thetrait. By measuring the appearance of the marker(s) in progeny ofcrosses, the existence of the trait can be detected by relatively simplemolecular tests without actually evaluating the appearance of the traititself, which can be difficult and time-consuming because the actualevaluation of the trait requires growing plants to a stage where thetrait can be expressed. Molecular markers have been widely used todetermine genetic composition in soybeans.

“Marker assisted selection” refers to the process of selecting a desiredtrait or traits in a plant or plants by detecting one or more nucleicacids from the plant, where the nucleic acid is linked to the desiredtrait, and then selecting the plant or germplasm possessing those one ormore nucleic acids.

The term “plant” includes reference to an immature or mature wholeplant, including a plant from which seed or grain or anthers have beenremoved. Seed or embryo that will produce the plant is also consideredto be the plant.

“Plant parts” means any portion or piece of a plant, including leaves,stems, buds, roots, root tips, anthers, seed, grain, embryo, pollen,ovules, flowers, cotyledons, hypocotyls, pods, flowers, shoots, stalks,tissues, tissue cultures, cells and the like.

“Polymorphism” means a change or difference between two related nucleicacids. A “nucleotide polymorphism” refers to a nucleotide that isdifferent in one sequence when compared to a related sequence when thetwo nucleic acids are aligned for maximal correspondence.

“Polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,”“nucleic acid fragment,” and “oligonucleotide” are used interchangeablyherein. These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural, oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more strands of cDNA, genomic DNA,synthetic DNA, or mixtures thereof.

“Primer” refers to an oligonucleotide (synthetic or occurringnaturally), which is capable of acting as a point of initiation ofnucleic acid synthesis or replication along a complementary strand whenplaced under conditions in which synthesis of a complementary strand iscatalyzed by a polymerase. Typically, primers are oligonucleotides from10 to 30 nucleic acids in length, but longer or shorter sequences can beemployed. Primers may be provided in double-stranded form, though thesingle-stranded form is preferred. A primer can further contain adetectable label, for example a 5′ end label.

“Probe” refers to an oligonucleotide (synthetic or occurring naturally)that is complementary (though not necessarily fully complementary) to apolynucleotide of interest and forms a duplexed structure byhybridization with at least one strand of the polynucleotide ofinterest. Typically, probes are oligonucleotides from 10 to 50 nucleicacids in length, but longer or shorter sequences can be employed. Aprobe can further contain a detectable label. The terms “label” and“detectable label” refer to a molecule capable of detection, including,but not limited to, radioactive isotopes, fluorescers, chemiluminescers,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals,ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like.A detectable label can also include a combination of a reporter and aquencher, such as are employed in FRET probes or TaqMan™ probes. Theterm “reporter” refers to a substance or a portion thereof which iscapable of exhibiting a detectable signal, which signal can besuppressed by a quencher. The detectable signal of the reporter is,e.g., fluorescence in the detectable range. The term “quencher” refersto a substance or portion thereof which is capable of suppressing,reducing, inhibiting, etc., the detectable signal produced by thereporter. As used herein, the terms “quenching” and “fluorescence energytransfer” refer to the process whereby, when a reporter and a quencherare in close proximity, and the reporter is excited by an energy source,a substantial portion of the energy of the excited state nonradiativelytransfers to the quencher where it either dissipates nonradiatively oris emitted at a different emission wavelength than that of the reporter.

“PRMMAT” means Predicted Relative Maturity. Soybean maturities aredivided into relative maturity groups. In the United States the mostcommon maturity groups are 00 through VIII. Within maturity groups 00through V are sub-groups. A sub-group is a tenth of a relative maturitygroup. Within narrow comparisons, the difference of a tenth of arelative maturity group equates very roughly to a day difference inmaturity at harvest.

“Rag genes,” “Rag intervals,” “Rag QTL,” and “Rag loci” refer to one ormore of the Rag1, Rag2, and Rag3 genes and the chromosome segments orintervals on which they are located. Rag1maps to linkage group M. Insome examples, the Rag1 interval is defined as being flanked by andincluding markers Satt540 and BARC-016783-02329. In other examples, theRag1 interval is defined as being flanked by and including markersBARC-039195-07466 and BARC-016783-02329. Rag2 maps to linkage group F.In some examples, the Rag2 interval is defined as being flanked by andincluding markers Satt334 and Sat_317. In other examples, the Rag2interval is defined as being flanked by and including markersBARC-029823-06424 and Sct_033. Rag3 maps to linkage group J. In someexamples, the Rag 3 interval is defined as being flanked by andincluding markers Sat_339 and Sct_065. In other examples, the Rag3interval is defined as being flanked by and including markersBARC-031195-07010 and Sat_370.

“Rag haplotype” or simply “haplotype” means the combination ofparticular alleles present within a particular plant's genome at one ormore specific marker loci within or linked to the Rag1, Rag2, or Rag3interval or gene. For instance, in one example, one specific SNP locuswithin or linked to the Rag1 interval is used to define a Rag1 haplotypefor a particular plant. In another example, two specific SNP loci withinor linked to the Rag1 interval are used to define a Rag1haplotype for aparticular plant. In still further examples, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more specific SNP loci withinor linked to the Rag1 interval are used to define a Rag1 haplotype for aparticular plant. The same applies for the Rag2 and Rag3 intervals.

In certain examples, multiple Rag haplotypes are used to define a“marker profile” or “Rag marker profile.” As used herein, “markerprofile” means the combination of two or more Rag haplotypes within aparticular plant's genome. For instance, in one example, a particularRag1 haplotype and a particular Rag2 haplotype define the marker profileof a particular plant. In another example, a particular Rag1 haplotypeand a particular Rag3 haplotype define the marker profile of aparticular plant. In a still further example, a particular Rag2haplotype and a particular Rag3 haplotype define the marker profile of aparticular plant. In an additional example, a particular Rag1 haplotype,a particular Rag2 haplotype, and a particular Rag3 haplotype define themarker profile of a particular plant. More specifically, a particularplant marker profile might be, for example, Rag1-a/Rag2-a orRag1-b/Rag2-a/Rag3-c.

“Recombination frequency” is the frequency of a crossing over event(recombination) between two genetic loci. Recombination frequency can beobserved by following the segregation of markers and/or traits duringmeiosis.

“Resistance” and “improved resistance” are used interchangeably hereinand refer to one or more of antibiosis resistance, antixenosisresistance, and tolerance to soybean aphid. “Antibiosis” refers to theplant's ability to reduce the survival, reproduction, and fecundity ofthe insect. “Antixenosis” refers to the plant's ability to deter theinsect from feeding or identifying the plant as a food source.“Tolerance” refers to the plant's ability to withstand heavy infestationwithout significant yield loss. A “resistant plant” or “resistant plantvariety” need not possess absolute or complete resistance to one or moresoybean aphid biotypes. Instead, a “resistant plant,” “resistant plantvariety,” or a plant or plant variety with “improved resistance” willhave a level of resistance to at least one soybean aphid biotype whichis higher than that of a comparable susceptible plant or variety.

“Self crossing,” “self pollination,” or “selfing” is a process throughwhich a breeder crosses a plant with itself; for example, a secondgeneration hybrid F2 with itself to yield progeny designated F2:3.

“SNP” or “single nucleotide polymorphism” means a sequence variationthat occurs when a single nucleotide (A, T, C, or G) in the genomesequence is altered or variable. “SNP markers” exist when SNPs aremapped to sites on the soybean genome. Many techniques for detectingSNPs are known in the art, including allele specific hybridization,primer extension, direct sequencing, and real-time PCR, such as theTaqMan™ assay.

“Stacking” refers to a process of producing a plant that has two or moreparticular alleles, haplotypes, or traits (generally desirable) thatwere received from different sources. For instance a plant with“stacked” alleles, may be produced by breeding two parental plants, eachof which has a different desirable allele, haplotype, or trait. Thestacked alleles, haplotypes, or traits may relate to the same phenotype,for instance alleles for different markers that both relate to aphidresistance, or may relate to different phenotypic traits, for instancestacking a favorable aphid resistance allele with a favorable droughtresistance allele. Additionally, stacking can involve two or morealleles/haplotypes at the same locus (e.g., Rag1a/Rag1b), or differentalleles/haplotypes at two or more distinct loci (e.g., Rag1a/Rag1b,Rag2a/Rag2b). Stacking can be done by any suitable method, includingthrough transgenic approaches or through breeding. In some instances,stacking is performed by using marker-assisted selection to selectparental plants with two or more desired alleles, haplotypes, or traitsand then crossing those plants to produce a stacked progeny plant.

“Transgenic plant” refers to a plant that comprises within its cells anexogenous polynucleotide. Generally, the exogenous polynucleotide isstably integrated within the genome such that the polynucleotide ispassed on to successive generations. The exogenous polynucleotide may beintegrated into the genome alone or as part of a recombinant expressioncassette. “Transgenic” is used herein to refer to any cell, cell line,callus, tissue, plant part, or plant, the genotype of which has beenaltered by the presence of exogenous nucleic acid including thosetransgenic organisms or cells initially so altered, as well as thosecreated by crosses or asexual propagation from the initial transgenicorganism or cell. The term “transgenic” as used herein does notencompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (e.g.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

The term “vector” is used in reference to polynucleotide or othermolecules that transfer nucleic acid segment(s) into a cell. A vectoroptionally comprises parts which mediate vector maintenance and enableits intended use (e.g., sequences necessary for replication, genesimparting drug or antibiotic resistance, a multiple cloning site,operably linked promoter/enhancer elements which enable the expressionof a cloned gene, etc.). Vectors are often derived from plasmids,bacteriophages, or plant or animal viruses.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofsoybean is commonly measured in bushels of seed per acre or metric tonsof seed per hectare per season. Yield is affected by both genetic andenvironmental factors. Yield is the final culmination of all agronomictraits.

Markers, Haplotypes, and Marker Profiles Associated with Resistance toSoybean Aphid:

Markers, primers, haplotypes, and marker profiles, and methods of theiruse for identifying and/or selecting soybean plants with improvedsoybean aphid resistance, are provided. The method for determining thepresence/absence/allele of a particular marker allele associated withsoybean aphid resistance and within or linked to a Rag gene or intervalin soybean plant or germplasm, and in turn determining the Rag haplotypeand/or marker profile of the plant/germplasm, comprises analyzinggenomic DNA from a soybean plant or germplasm to determine if at leastone, or a plurality, of such markers is present or absent and in whatallelic form. Using this information regarding the Rag-associatedmarkers present in the particular plant or germplasm in turn allows aRag haplotype to be assigned to that plant/germplasm. If multiple Raghaplotypes are deduced for a plant, a marker profile can in turn beassigned by combining all of these Rag haplotypes.

In certain examples, plants or germplasm are identified that have atleast one favorable allele, haplotype, or marker profile that positivelycorrelates with resistance or improved resistance. However, in otherexamples, it is useful for exclusionary purposes during breeding toidentify alleles, haplotypes, or marker profiles that negativelycorrelate with resistance, for example to eliminate such plants orgermplasm from subsequent rounds of breeding.

While any marker linked to a Rag gene or interval is useful, markersthat map closer to a Rag gene or interval are generally preferred overmarkers that map farther from a Rag gene or interval. Marker loci areespecially useful when they are closely linked to a Rag gene orinterval. Thus, in one example, marker loci display an inter-locuscross-over frequency of about 10% or less, about 9% or less, about 8% orless, about 7% or less, about 6% or less, about 5% or less, about 4% orless, about 3% or less, about 2% or less, about 1% or less, about 0.75%or less, about 0.5% or less, or about 0.25% or less with the Rag gene towhich they are linked. Thus, the loci are separated from the Rag gene towhich they are linked by about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM, or 0.25 cM or less.

In certain examples, multiple marker loci that collectively make up theRag haplotype of interest are investigated, for instance 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more marker loci.

In certain examples, markers useful for defining a Rag1 haplotype arelinked or are closely linked to the interval flanked by and includingthe marker loci Satt540 and BARC-016783-02329 in the Soybase database(soybase.org). In other examples, markers useful for defining a Rag1haplotype are linked or are closely linked to the interval flanked byand including the marker loci BARC-039195-07466 and BARC-016783-02329 inthe Soybase database (soybase.org). In still further examples, markersuseful for defining a Rag1 haplotype are within the interval flanked byand including Satt540 and BARC-016783-02329 or BARC-039195-07466 andBARC-016783-02329 in the Soybase database (soybase.org). In otherparticular examples, the markers useful for defining a Rag1 haplotypeare within the interval flanked by and including Satt435 and Sat_244 inthe Soybase database (soybase.org). In further particular examples, themarkers useful for defining a Rag1 haplotype are within the intervalflanked by and including physical position 5453161-8194502 on LG-M onthe Glymal soybean genome assembly.

In additional examples, markers useful for defining a Rag2 haplotype arelinked or are closely linked to the interval flanked by and includingthe marker loci Satt334 and Sat_317 in the Soybase database(soybase.org). In other examples, markers useful for defining a Rag2haplotype are linked to or are closely linked to the interval flanked byand including the marker loci BARC-029823-06424 and Sct_033 in theSoybase database (soybase.org). In still further examples, markersuseful for defining a Rag2 haplotype are within the interval flanked byand including Satt334 and Sat_317 or BARC-029823-06424 and Sct_033 inthe Soybase database (soybase.org). In other particular examples, themarkers useful for defining a Rag2 haplotype are within the intervalflanked by and including Satt334 and Satt510 in the Soybase database(soybase.org). In further particular examples, the markers useful fordefining a Rag2 haplotype are within the interval flanked by andincluding physical position 28416122-30590233 on LG-F on the Glymalsoybean genome assembly.

In yet further examples, markers useful for defining a Rag3 haplotypeare linked or are closely linked to the interval flanked by andincluding the marker loci Sat_339 and Sct_065 in the Soybase database(soybase.org). In still further examples, markers useful for defining aRag3 haplotype are linked or are closely linked to the interval flankedby and including the marker loci BARC-031195-07010 and Sat_370 in theSoybase database (soybase.org). In still further examples, markersuseful for defining a Rag3 haplotype are within the interval flanked byand including Sat_339 and Sct_065 or BARC-031195-07010 and Sat_370 inthe Soybase database (soybase.org). In other particular examples, themarkers useful for defining a Rag3 haplotype are within the intervalflanked by and including Sat_339 and Sat_370 in the Soybase database(soybase.org). In further particular examples, the markers useful fordefining a Rag3 haplotype are within the interval flanked by andincluding physical position 4157916-7054678 on LG-J on the Glymalsoybean genome assembly.

Markers within, linked to, or closely linked to these intervals areillustrated in the genetic map of FIG. 1. Numerous such markers are alsowell known in the art, for example, are described in the USDA's soybasedatabase, available at www.soybase.org.

Exemplary markers useful for defining Rag haplotypes are provided inTable 1. Also provided in Table 1 are the target regions containing themarkers, as well as primers and probes that can be used to amplify anddetect the markers.

In certain examples the marker loci used to define the Rag1 haplotypeare one or more of S15354-001-Q001, S14181-1-Q1, S13871-1-Q1,S14161-1-Q10, S09515-1-Q1, S14151-1-Q1, S14151-2-Q4, S07164-1-Q12,S14182-1-Q1, S00812-1-A, and S02780-1-A. In other examples, the markerloci used to define the Rag1 haplotype are two or more ofS15354-001-Q001, S14181-1-Q1, S13871-1-Q1, S14161-1-Q10, S09515-1-Q1,S14151-1-Q1, S14151-2-Q4, S07164-1-Q12, S14182-1-Q1, S00812-1-A, andS02780-1-A. In further examples, the marker loci used to define the Rag1haplotype are three or more of S15354-001-Q001, S14181-1-Q1,S13871-1-Q1, S14161-1-Q10, S09515-1-Q1, S14151-1-Q1, S14151-2-Q4,S07164-1-Q12, S14182-1-Q1, S00812-1-A, and S02780-1-A. In additionalexamples, the marker loci used to define the Rag1 haplotype are four ormore of S15354-001-Q001, S14181-1-Q1, S13871-1-Q1, S14161-1-Q10,S09515-1-Q1, S14151-1-Q1, S14151-2-Q4, S07164-1-Q12, S14182-1-Q1,S00812-1-A, and S02780-1-A. In still further examples, the marker lociused to define the Rag1 haplotype are five or more of S15354-001-Q001,S14181-1-Q1, S13871-1-Q1, S14161-1-Q10, S09515-1-Q1, S14151-1-Q1,S14151-2-Q4, S07164-1-Q12, S14182-1-Q1, S00812-1-A, and S02780-1-A. Inyet further examples, the marker loci used to define the Rag1 haplotypeare all of S15354-001-Q001, S14181-1-Q1, S13871-1-Q1, S14161-1-Q10,S09515-1-Q1, S14151-1-Q1, S14151-2-Q4, S07164-1-Q12, S14182-1-Q1,S00812-1-A, and S02780-1-A. In a particular example, the marker lociused to define the Rag1 haplotype are all of S14161-1-Q10, S09515-1-Q1,S14151-2-Q4, and S07164-1-Q12.

In certain examples, the marker loci used to define the Rag2 haplotypeare one or more of S01190-1-A, S14761-001-Q001, S14771-001-Q001,S07165-1-Q3, S14778-001-Q001, and S01164-1-Q1. In other examples, themarker loci used to define the Rag2 haplotype are two or more ofS01190-1-A, S14761-001-Q001, S14771-001-Q001, S07165-1-Q3,S14778-001-Q001, and S01164-1-Q1. In additional examples, the markerloci used to define the Rag2 haplotype are three or more of S01190-1-A,S14761-001-Q001, S14771-001-Q001, S07165-1-Q3, S14778-001-Q001, andS01164-1-Q1. In further examples, the marker loci used to define theRag2 haplotype are four or more of S01190-1-A, S14761-001-Q001,S14771-001-Q001, S07165-1-Q3, S14778-001-Q001, and S01164-1-Q1. In stillfurther examples, the marker loci used to define the Rag2 haplotype areall of S14761-001-Q001, S14771-001-Q001, S07165-1-Q3, S14778-001-Q001,and S01164-1-Q1. In a particular example, the marker loci used to definethe Rag2 haplotype are S07165-1-Q3, S01190-1-A, and S01164-1-Q1.

In certain examples, the marker loci used to define the Rag3 haplotypeare one or more of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.In other examples, the marker loci used to define the Rag3 haplotype aretwo or more of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.In additional examples, the marker loci used to define the Rag3haplotype are three or more of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.In further examples, the marker loci used to define the Rag3 haplotypeare four or more of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.In still further examples, the marker loci used to define the Rag3haplotype are five or more of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1,S13664-1-Q1/Q002, S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1.examples, the marker loci used to define the Rag3 haplotype are one ormore of S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1, S13664-1-Q1/Q002,S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1. In additionalexamples, the marker loci used to define the Rag3 haplotype are all ofS13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1, S13664-1-Q1/Q002,S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, and S13675-2-Q1. In a particularexample, the marker loci used to define the Rag3 haplotype are all ofS11411-1-Q1, S13674-1-Q1/Q007, and S13675-2-Q1.

TABLE 1Selected markers useful for defining Rag haplotypes and marker profilesComposite Map Physical Probes (probe1-FAM/ Position pos. of Geno-probe2-VIC; SNP base (cM) SNP types Marker Forward and Reverse Primersindicated by capital letter) Rag1 (LG-M) 25.7 cM 5453161 A/T S15354-SEQ ID NO: 132 SEQ ID NO: 134 001-Q1 ccaaatcagatatcaagatgatggtatctatgAttccattttt SEQ ID NO: 133 SEQ ID NO: 135 ggccaagcaatacacaagaaaatcatctagTttccatttt 26.06 cM 5516385 A/G S14181- SEQ ID: 1 SEQ ID: 31-Q1 gcatctcatgattaagtagg caatcaGcacccttg SEQ ID: 2 SEQ ID: 4caagaactttgcttgtcttgctg aagcaatcaAcaccctt 26.06 cM 5516818 C/T S13871-SEQ ID: 6 SEQ ID: 8 1-Q1 gcaggctcatcagattgctt ttgaaacCaccatttt SEQ ID: 7SEQ ID: 9 gcagcgtctcatcaacaaaa aaacTaccattttgc 26.49 cM 5598980 C/GS14161- SEQ ID: 11 SEQ ID: 13 1-Q10 caccagctcgataagctagagatccagtagcaGcccta SEQ ID: 12 SEQ ID: 14 ttagccatggattttgttgaatacagtagcaCccctaccaa 26.51 cM 5602544 A/G S09515- SEQ ID: 16 SEQ ID: 181-Q1 tgcaagattgatttttatgatacgg tattgccaAttcgatcc SEQ ID: 17 SEQ ID: 19ggactaaaattagaaaaagaggaacca tattgccaGttcgatc 26.52 cM 5605203 A/CS14151- SEQ ID: 21 SEQ ID: 23 1-Q1 ccagcttcttttgctccatc cattgtacgTccctcSEQ ID: 22 SEQ ID: 24 cgacgctcctaagtattggtg atcattgtacgGccc 26.52 cM5605275 A/G S14151- SEQ ID: 26 SEQ ID: 28 2-Q4 aatcccacaccagcttcttttcagaacaTcttggc SEQ ID: 27 SEQ ID: 29 gtgtggcactgtagcagataaagatacagaacaCcttggc 26.54 cM 5608106 A/G S07164- SEQ ID: 31 SEQ ID: 33 1-Q12tcatttcctgatgctcaccata ttgagaaaacGtctca SEQ ID: 32 SEQ ID: 34ggttgtatccatcttctgaactgc ttgagaaaacAtctgca 26.6 cM 5630404 A/G S14182-SEQ ID: 36 SEQ ID: 38 1-Q1 tgtactttggctgcgtctcc ccatgtcaaTgcc SEQ ID: 37SEQ ID: 39 ggtaactcctttgtaatgttcaccac ccatgtcaaCgcca 33.2 cM 6754454 C/GS00812- SEQ ID: 41 SEQ ID: 43 1-A gctgctctttctctgctgtgatcatataccCgtgagactat SEQ ID: 42 SEQ ID: 44 tgggtggtttccttgtttataccaactataccGgtgagactat 34.2 cM 6671535 A/G S02780- SEQ ID: 46 SEQ ID: 48 1-Aggcatttgcttcaattttcc actctggAtaacctg SEQ ID: 47 SEQ ID: 49acttttgcccctatakgatatgc actctggGtaacctg Rag2 (LG-F) 70.08 28187733 A/TS01190- SEQ ID: 127 SEQ ID: 129 1-A ttcagctccccattatttcg tcagctcaTttttgtSEQ ID: 128 SEQ ID: 130 ttggccaacctatcctcaac cagctcaCttttgt 72.8528829625 A/G S14761- SEQ ID: 51 SEQ ID: 53 001-agagagcaacaaccagtaatttcata ccactaaAgttagcctag Q001 SEQ ID: 52 SEQ ID: 54acttagtgcatctattgcaaccac ccactaaGgttagcctag 72.85 28837383 C/T S14771-SEQ ID: 56 SEQ ID: 58 001- ccttcaacaacagcagctttaat cattagatcaaacaCtgcQ001 SEQ ID: 57 SEQ ID: 59 ctgcttaatcgactgagctagacc cattagatcaaacaTtgc73.0 cM 29097652 A/T S07165- SEQ ID: 61 SEQ ID: 63 1-Q3gcttgtaagctattcccaaacg tttcttatcTaaggttttg SEQ ID: 62 SEQ ID: 64tatctgtgagcggttgcttg ttcttatcAaaggttttg 73.2 29678319 C/T S14778-SEQ ID: 66 SEQ ID: 68 001- tgaggatatttatggaatttgtcaga cttataaaacCgctttcQ001 SEQ ID: 67 SEQ ID: 69 catgatgagatcagaaaagaaatgc cttataaaacTgctttcc73.2 cM 29825175 C/G S01164- SEQ ID: 71 SEQ ID: 73 1-Q1gacagtggagagttacgagga ccacctacatCactac SEQ ID: 72 SEQ ID: 74cacatctgaatcaccctgga ccacctacatGactac Rag3 (JG-J) 37.8800 5140274 A/GS13662- SEQ ID: 76 SEQ ID: 78 1-Q3 tctttatgatgatgagcagaagctactttcagAgcattagc SEQ ID: 77 SEQ ID: 79 caccccaaaaacaaaacactctttgctttcagGgcat S13662- SEQ ID: 80 SEQ ID: 82 1-Q6gggaagagtctgaatggtgtct ctttcagAgcattagc SEQ ID: 81 SEQ ID: 83ccccaaaaacaaaacactcatc tttgctttcagGgcat 41.7323 5919650 T/C S13663-SEQ ID: 85 SEQ ID: 87 1-Q1 tctgatgatgattatagtgggctct ctgataacaaTagcccSEQ ID: 86 SEQ ID: 88 tgctatgcatttgaaaccaca ataacaaCagccctgact 41.965960726 C/G S11411- SEQ ID: 90 SEQ ID: 92 1-Q1 ggacccaacatcaatcaaatgttttctgCactccc SEQ ID: 91 SEQ ID: 93 tgcattctggaaagacatgg ttttctgGactccc42.5533 6066531 T/G S13664- SEQ ID: 95 SEQ ID: 97 1-Q1catgccagtatgaatgtgctg attgtgacactctatTgc SEQ ID: 96 SEQ ID: 98tccgcacatttagttccctta ttgtgacactctatGgca S13664- SEQ ID: 99 SEQ ID: 1011-Q002 caaagtgtcatgccagtatgaatg attgtgacactctatTgc SEQ ID: 100SEQ ID: 102 gttttattttcattccgcacatttag ttgtgacactctatGgca 42.97576231641 G/A S13672- SEQ ID: 104 SEQ ID: 106 1-Q1 gatcggttcccaaactagcacagttgattactCtgc SEQ ID: 105 SEQ ID: 107 aacatgcaaaatgcaccaagcagttgattactTtgc S13672- SEQ ID: 108 SEQ ID: 106 1-Q2cggttcccaaactagcaggt cagttgattactCtgc SEQ ID: 109 SEQ ID: 107tgcaaaatgcaccaagttagat cagttgattactTtgc S13672- SEQ ID: 110 SEQ ID: 1121-Q3 agatcggttcccaaactagcag cagttgattactCtgc SEQ ID: 111 SEQ ID: 112catgcaaaatgcaccaagtta cagttgattactTtgc 43.7295 6524877 C/G S13674-SEQ ID: 115 SEQ ID: 117 1-Q1 ccaccattacccctctcctt ttggcattcaGcccSEQ ID: 116 SEQ ID: 118 acctagcattgcaatctcttcc tttggcattcaCccc S13674-SEQ ID: 119 SEQ ID: 117 1-Q007 ttacccctctcctttctcaacatta ttggcattcaGcccSEQ ID: 120 SEQ ID: 118 tgcaatctcttccaagctagaact tttggcattcaCccc 43.81866542422 G/A S13675- SEQ ID: 122 SEQ ID: 124 2-Q1 aggtggtggcagtgttgattaaccgtggctCatt SEQ ID: 123 SEQ ID: 125 ctccaacatggctgtgctaacaaaccgtggctTat

Selected haplotypes that are based upon the markers in Table 1 aredescribed in Table 2.

TABLE 2 Selected Rag haplotypes generated using the selected markers RagHaplotypes Rag1 S14161-1- S14151-2- S07164-1- Q10 S09515-1-Q1 Q4 Q12Haplotype C G G G Rag1-a G A G A Rag1-b C, G A A A Rag1-c C G A A Rag1-dC A A A Rag1-e G A A A Rag1-f C A, G G A Rag1-g Rag2 S07165-1- S01190-1-S01164-1- Q3 A Q1 Haplotype A C C Rag2-a T T G Rag2-b T C C Rag2-c A T GRag2-d T C G Rag2-f Rag3 S11411-1- S13674-1- S13675-2- Q1 Q1/Q007 Q1Haplotype C C G Rag3-a G C G Rag3-b G G A Rag3-c G G G Rag3-d

In addition to the markers discussed herein, information regardinguseful soybean markers can be found, for example, on the USDA's Soybasewebsite, available at www.soybase.org. One of skill in the art willrecognize that the identification of favorable marker alleles may begermplasm-specific. The determination of which marker alleles correlatewith resistance (or susceptibility) is determined for the particulargermplasm under study. One of skill will also recognize that methods foridentifying the favorable alleles are routine and well known in the art,and furthermore, that the identification and use of such favorablealleles is well within the scope of the invention.

In some examples marker profiles comprising two or more Rag haplotypesare provided. For instance, in one example, a particular Rag1 haplotypeand a particular Rag2 haplotype define the marker profile of aparticular plant. In another example, a particular Rag1 haplotype and aparticular Rag3 haplotype define the marker profile of a particularplant. In a still further example, a particular Rag2 haplotype and aparticular Rag3 haplotype define the marker profile of a particularplant. In an additional example, a particular Rag1 haplotype, aparticular Rag2 haplotype, and a particular Rag3 haplotype define themarker profile of a particular plant. More specifically, a particularplant marker profile might be, for example, Rag1-a/Rag2-b orRag1-b/Rag2-a/Rag3-c.

Marker Assisted Selection:

The use of marker assisted selection (MAS) to select a soybean plant orgermplasm which has a certain Rag haplotype or marker profile isprovided. For instance, in certain examples a soybean plant or germplasmpossessing a certain predetermined favorable Rag haplotype will beselected via MAS. In certain other examples, a soybean plant orgermplasm possessing a certain predetermined favorable marker profilewill be selected via MAS.

Using MAS, soybean plants or germplasm can be selected for markers ormarker alleles that positively correlate with resistance, withoutactually raising soybean and measuring for resistance or improvedresistance (or, contrawise, soybean plants can be selected against ifthey possess markers that negatively correlate with resistance orimproved resistance). MAS is a powerful tool to select for desiredphenotypes and for introgressing desired traits into cultivars ofsoybean (e.g., introgressing desired traits into elite lines). MAS iseasily adapted to high throughput molecular analysis methods that canquickly screen large numbers of plant or germplasm genetic material forthe markers of interest and is much more cost effective than raising andobserving plants for visible traits.

Nucleic Acid Amplification Methods:

In some examples, the molecular markers are detected using a suitableamplification-based detection method. In these types of methods, nucleicacid primers are typically hybridized to the conserved regions flankingthe polymorphic marker region. In certain methods, nucleic acid probesthat bind to the amplified region are also employed. In general,synthetic methods for making oligonucleotides, including primers andprobes, are well known in the art. For example, oligonucleotides can besynthesized chemically according to the solid phase phosphoramiditetriester method described by Beaucage and Caruthers (1981) TetrahedronLetts 22:1859-1862, e.g., using a commercially available automatedsynthesizer, e.g., as described in Needham-VanDevanter, et al. (1984)Nucleic Acids Res. 12:6159-6168. Oligonucleotides, including modifiedoligonucleotides, can also be ordered from a variety of commercialsources known to persons of skill in the art.

It will be appreciated that suitable primers and probes to be used canbe designed using any suitable method. It is not intended that theinvention be limited to any particular primer, primer pair or probe. Forexample, primers can be designed using any suitable software program,such as LASERGENE® or Primer3.

It is not intended that the primers be limited to generating an ampliconof any particular size. For example, the primers used to amplify themarker loci and alleles herein are not limited to amplifying the entireregion of the relevant locus. In some examples, marker amplificationproduces an amplicon at least 20 nucleotides in length, oralternatively, at least 50 nucleotides in length, or alternatively, atleast 100 nucleotides in length, or alternatively, at least 200nucleotides in length.

PCR, RT-PCR, and LCR are in particularly broad use as amplification andamplification-detection methods for amplifying nucleic acids of interest(e.g., those comprising marker loci), facilitating detection of themarkers. Details regarding the use of these and other amplificationmethods are well known in the art and can be found in any of a varietyof standard texts. Details for these techniques can also be found innumerous journal and patent references, such as Mullis, et al. (1987)U.S. Pat. No. 4,683,202; Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47;Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli, etal., (1990) Proc. Natl. Acad. Sci. USA87:1874; Lomell, et al., (1989) J.Clin. Chem 35:1826; Landegren, et al., (1988) Science 241:1077-1080; VanBrunt, (1990) Biotechnology 8:291-294; Wu and Wallace, (1989) Gene4:560; Barringer, et al., (1990) Gene 89:117, and Sooknanan and Malek,(1995) Biotechnology 13:563-564.

Such nucleic acid amplification techniques can be applied to amplifyand/or detect nucleic acids of interest, such as nucleic acidscomprising marker loci. Amplification primers for amplifying usefulmarker loci and suitable probes to detect useful marker loci or togenotype SNP alleles are provided. For example, exemplary primers andprobes are provided in Table 1, as are the target regions to which theseprimers and probes hybridize. However, one of skill will immediatelyrecognize that other primer and probe sequences could also be used. Forinstance primers to either side of the given primers can be used inplace of the given primers, so long as the primers can amplify a regionthat includes the allele to be detected, as can primers and probesdirected to other SNP marker loci. Further, it will be appreciated thatthe precise probe to be used for detection can vary, e.g., any probethat can identify the region of a marker amplicon to be detected can besubstituted for those examples provided herein. Further, theconfiguration of the amplification primers and detection probes can, ofcourse, vary. Thus, the compositions and methods are not limited to theprimers and probes specifically recited herein.

In certain examples, probes will possess a detectable label. Anysuitable label can be used with a probe. Detectable labels suitable foruse with nucleic acid probes include, for example, any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means. Useful labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetriclabels. Other labels include ligands, which bind to antibodies labeledwith fluorophores, chemiluminescent agents, and enzymes. A probe canalso constitute radiolabelled PCR primers that are used to generate aradiolabelled amplicon. Labeling strategies for labeling nucleic acidsand corresponding detection strategies can be found, e.g., in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals SixthEdition by Molecular Probes, Inc. (Eugene Oreg.); or Haugland (2001)Handbook of Fluorescent Probes and Research Chemicals Eighth Edition byMolecular Probes, Inc. (Eugene Oreg.).

Detectable labels may also include reporter-quencher pairs, such as areemployed in Molecular Beacon and TaqMan™ probes. The reporter may be afluorescent organic dye modified with a suitable linking group forattachment to the oligonucleotide, such as to the terminal 3′ carbon orterminal 5′ carbon. The quencher may also be an organic dye, which mayor may not be fluorescent, depending on the embodiment. Generally,whether the quencher is fluorescent or simply releases the transferredenergy from the reporter by non-radiative decay, the absorption band ofthe quencher should at least substantially overlap the fluorescentemission band of the reporter to optimize the quenching. Non-fluorescentquenchers or dark quenchers typically function by absorbing energy fromexcited reporters, but do not release the energy radiatively.

Selection of appropriate reporter-quencher pairs for particular probesmay be undertaken in accordance with known techniques. Fluorescent anddark quenchers and their relevant optical properties from whichexemplary reporter-quencher pairs may be selected are listed anddescribed, for example, in Berlman, Handbook of Fluorescence Spectra ofAromatic Molecules, 2nd ed., Academic Press, New York, 1971, the contentof which is incorporated herein by reference. Examples of modifyingreporters and quenchers for covalent attachment via common reactivegroups that can be added to an oligonucleotide in the present inventionmay be found, for example, in Haugland, Handbook of Fluorescent Probesand Research Chemicals, Molecular Probes of Eugene, Oreg., 1992, thecontent of which is incorporated herein by reference.

In certain examples, reporter-quencher pairs are selected from xanthenedyes including fluoresceins and rhodamine dyes. Many suitable forms ofthese compounds are available commercially with substituents on thephenyl groups, which can be used as the site for bonding or as thebonding functionality for attachment to an oligonucleotide. Anotheruseful group of fluorescent compounds for use as reporters are thenaphthylamines, having an amino group in the alpha or beta position.Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5 sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include3-phenyl-7-isocyanatocoumarin; acridines such as9-isothiocyanatoacridine; N-(p-(2-b enzoxazolyl)phenyl)maleimide;benzoxadiazoles; stilbenes; pyrenes and the like. In certain otherexamples, the reporters and quenchers are selected from fluorescein andrhodamine dyes. These dyes and appropriate linking methodologies forattachment to oligonucleotides are well known in the art.

Suitable examples of reporters may be selected from dyes such as SYBRgreen, 5-carboxyfluorescein (5-FAM™ available from Applied Biosystems ofFoster City, Calif.), 6-carboxyfluorescein (6-FAM),tetrachloro-6-carboxyfluorescein (TET),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein,hexachloro-6-carboxyfluorescein (HEX), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TET™ available from AppliedBiosystems), carboxy-X-rhodamine (ROX), 6-carboxy-4′, 5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOE™ available from Applied Biosystems), VIC™dye products available from Molecular Probes, Inc., NED™ dye productsavailable from available from Applied Biosystems, and the like. Suitableexamples of quenchers may be selected from6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo) benzoicacid (DABYL), tetramethylrhodamine (TAMRA), BHQ-0™, BHQ-1™, BHQ-2™, andBHQ-3™, each of which are available from Biosearch Technologies, Inc. ofNovato, Calif., QSY-7™, QSY-9198 , QSY-21™ and QSY-35™, each of whichare available from Molecular Probes, Inc., and the like.

In one aspect, real time PCR or LCR is performed on the amplificationmixtures described herein, e.g., using molecular beacons or TaqMan™probes. A molecular beacon (MB) is an oligonucleotide which, underappropriate hybridization conditions, self-hybridizes to form a stem andloop structure. The MB has a label and a quencher at the termini of theoligonucleotide; thus, under conditions that permit intra-molecularhybridization, the label is typically quenched (or at least altered inits fluorescence) by the quencher. Under conditions where the MB doesnot display intra-molecular hybridization (e.g., when bound to a targetnucleic acid, such as to a region of an amplicon during amplification),the MB label is unquenched. Details regarding standard methods of makingand using MBs are well established in the literature and MBs areavailable from a number of commercial reagent sources. See also, e.g.,Leone, et al., (1995) Molecular beacon probes combined withamplification by NASBA enable homogenous real-time detection of RNA,Nucleic Acids Res. 26:2150-2155; Tyagi and Kramer, (1996) Molecularbeacons: probes that fluoresce upon hybridization, Nature Biotechnology14:303-308; Blok and Kramer, (1997) Amplifiable hybridization probescontaining a molecular switch, Mol Cell Probes 11:187-194; Hsuih. etal., (1997) Novel, ligation-dependent PCR assay for detection ofhepatitis C in serum, J Clin Microbiol 34:501-507; Kostrikis, et al.,(1998) Molecular beacons: spectral genotyping of human alleles, Science279:1228-1229; Sokol, et al., (1998) Real time detection of DNA:RNAhybridization in living cells, Proc. Natl. Acad. Sci. U.S.A.95:11538-11543; Tyagi, et al., (1998) Multicolor molecular beacons forallele discrimination, Nature Biotechnology 16:49-53; Bonnet, et al.,(1999) Thermodynamic basis of the chemical specificity of structured DNAprobes, Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang, et al. (1999)Designing a novel molecular beacon for surface-immobilized DNAhybridization studies, J. Am. Chem. Soc. 121:2921-2922; Marras, et al.,(1999) Multiplex detection of single-nucleotide variation usingmolecular beacons, Genet. Anal. Biomol. Eng. 14:151-156; and Vet, etal., (1999) Multiplex detection of four pathogenic retroviruses usingmolecular beacons, Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399.Additional details regarding MB construction and use is found in thepatent literature, e.g., U.S. Pat. Nos. 5,925,517; 6,150,097; and6,037,130.

Another real-time detection method is the 5′-exonuclease detectionmethod, also called the TaqMan™ assay, as set forth in U.S. Pat. Nos.5,804,375; 5,538,848; 5,487,972; and 5,210,015, each of which is herebyincorporated by reference in its entirety. In the TaqMan™ assay, amodified probe, typically 10-25 nucleic acids in length, is employedduring PCR which binds intermediate to or between the two members of theamplification primer pair. The modified probe possesses a reporter and aquencher and is designed to generate a detectable signal to indicatethat it has hybridized with the target nucleic acid sequence during PCR.As long as both the reporter and the quencher are on the probe, thequencher stops the reporter from emitting a detectable signal. However,as the polymerase extends the primer during amplification, the intrinsic5′ to 3′ nuclease activity of the polymerase degrades the probe,separating the reporter from the quencher, and enabling the detectablesignal to be emitted. Generally, the amount of detectable signalgenerated during the amplification cycle is proportional to the amountof product generated in each cycle.

It is well known that the efficiency of quenching is a strong functionof the proximity of the reporter and the quencher, i.e., as the twomolecules get closer, the quenching efficiency increases. As quenchingis strongly dependent on the physical proximity of the reporter andquencher, the reporter and the quencher are preferably attached to theprobe within a few nucleotides of one another, usually within 30nucleotides of one another, more preferably with a separation of fromabout 6 to 16 nucleotides. Typically, this separation is achieved byattaching one member of a reporter-quencher pair to the 5′ end of theprobe and the other member to a nucleotide about 6 to 16 nucleotidesaway, in some cases at the 3′ end of the probe.

Separate detection probes can also be omitted in amplification/detectionmethods, e.g., by performing a real time amplification reaction thatdetects product formation by modification of the relevant amplificationprimer upon incorporation into a product, incorporation of labelednucleotides into an amplicon, or by monitoring changes in molecularrotation properties of amplicons as compared to unamplified precursors(e.g., by fluorescence polarization).

Further, it will be appreciated that amplification is not a requirementfor marker detection—for example, one can directly detect unamplifiedgenomic DNA simply by performing a Southern blot on a sample of genomicDNA. Procedures for performing Southern blotting, amplification e.g.,(PCR, LCR, or the like), and many other nucleic acid detection methodsare well established and are taught, e.g., in Sambrook, et al.,Molecular Cloning—A Laboratory Manual (3d ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); CurrentProtocols in Molecular Biology, F. M. Ausubel, et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2002) (“Ausubel”))and PCR Protocols A Guide to Methods and Applications (Innis, et al.,eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Additionaldetails regarding detection of nucleic acids in plants can also befound, e.g., in Plant Molecular Biology (1993) Croy (ed.) BIOSScientific Publishers, Inc.

Other techniques for detecting SNPs can also be employed, such as allelespecific hybridization (ASH). ASH technology is based on the stableannealing of a short, single-stranded, oligonucleotide probe to acompletely complementary single-stranded target nucleic acid. Detectionis via an isotopic or non-isotopic label attached to the probe. For eachpolymorphism, two or more different ASH probes are designed to haveidentical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization.

Real-Time SNP Detection Assays:

Real-time amplification assays, including MB or TaqMan™ based assays,are especially useful for detecting SNP alleles. In such cases, probesare typically designed to bind to the amplicon region that includes theSNP locus, with one allele-specific probe being designed for eachpossible SNP allele. For instance, if there are two known SNP allelesfor a particular SNP locus, “A” or “C,” then one probe is designed withan “A” at the SNP position, while a separate probe is designed with a“C” at the SNP position. While the probes are typically identical to oneanother other than at the SNP position, they need not be. For instance,the two allele-specific probes could be shifted upstream or downstreamrelative to one another by one or more bases. However, if the probes arenot otherwise identical, they should be designed such that they bindwith approximately equal efficiencies, which can be accomplished bydesigning under a strict set of parameters that restrict the chemicalproperties of the probes. Further, a different detectable label, forinstance a different reporter-quencher pair, is typically employed oneach different allele-specific probe to permit differential detection ofeach probe. In certain examples, each allele-specific probe for acertain SNP locus is 11-20 nucleotides in length, dual-labeled with aflorescence quencher at the 3′ end and either the 6-FAM(6-carboxyfluorescein) or VIC(4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein) fluorophore at the 5′end.

To effectuate SNP allele detection, a real-time PCR reaction can beperformed using primers that amplify the region including the SNP locus,for instance the target regions listed in Table 1, the reaction beingperformed in the presence of all allele-specific probes for the givenSNP locus. By then detecting signal for each detectable label employedand determining which detectable label(s) demonstrated an increasedsignal, a determination can be made of which allele-specific probe(s)bound to the amplicon and, thus, which SNP allele(s) the ampliconpossessed. For instance, when 6-FAM- and VIC-labeled probes areemployed, the distinct emission wavelengths of 6-FAM (518 nm) and VIC(554 nm) can be captured. A sample that is homozygous for one allelewill have fluorescence from only the respective 6-FAM or VICfluorophore, while a sample that is heterozygous at the analyzed locuswill have both 6-FAM and VIC fluorescence.

The KASPar® and Illumina® Detection Systems are additional examples ofcommercially-available marker detection systems. KASPar® is ahomogeneous fluorescent genotyping system which utilizes allele specifichybridization and a unique form of allele specific PCR (primerextension) in order to identify genetic markers (e.g. a particular SNPlocus associated with aphid resistance). Illumina® detection systemsutilize similar technology in a fixed platform format. The fixedplatform utilizes a physical plate that can be created with up to 384markers. The Illumina® system is created with a single set of markersthat cannot be changed and utilizes dyes to indicate marker detection.

These systems and methods represent a wide variety of availabledetection methods which can be utilized to detect markers associatedwith improved aphid resistance, but any other suitable method could alsobe used.

Stacking Desired Rag Alleles, Haplotypes, or Marker Profiles:

Methods of stacking desired Rag alleles, haplotypes, or marker profilesare provided, as are methods of detecting soybean plants or germplasmthat possess stacked alleles, haplotypes, or marker profiles. In orderto stack desired alleles, haplotypes, or marker profiles, two or moreplants with different desired alleles, haplotypes, or marker profilesare first identified. Following identification of the desired alleles,haplotypes, or marker profiles, any method for introgressing an allele,haplotype, or marker profile into soybean plants known to one of skillin the art can be used, including breeding and transgenic methods.Typically, a first soybean germplasm that contains a desired allele,haplotype, or marker profile and a second soybean germplasm thatcontains a different desired allele, haplotype, or marker profile areprovided. The first soybean germplasm may be crossed with the secondsoybean germplasm to provide progeny soybean germplasm. These progenygermplasm are screened to determine the presence of the desiredphenotype and or the presence of both of the desired alleles,haplotypes, or marker profiles, and progeny that test positive for thepresence of the desired alleles, haplotypes, or marker profiles areselected. Methods for performing such screening are well known in theart and any suitable method can be used. In certain examples, thestacked plants can also be self-crossed in order to generatehomozygosity.

Phenotypic Screening for Soybean Aphid Resistant Soybean Plants:

Three types of soybean aphid resistance have been described: antibiosis,antixenosis, and tolerance. Experienced plant breeders can recognizeresistant soybean plants in the field, and can select the resistantindividuals or populations for breeding purposes or for propagation. Inthis context, the plant breeder recognizes “resistant” and“non-resistant” or “susceptible” soybean plants. However, plantresistance is a phenotypic spectrum consisting of extremes in resistanceand susceptibility, as well as a continuum of intermediate resistancephenotypes. Evaluation of these intermediate phenotypes usingreproducible assays are of value to scientists who seek to identifygenetic loci that impart resistance, to conduct marker assistedselection for resistance populations, and to use introgressiontechniques to breed a resistance trait into an elite soybean line, forexample.

To that end, screening and selection of resistant soybean plants may beperformed, for example, by exposing plants to soybean aphid in a liveaphid assay and selecting those plants showing resistance to aphids. Thelive aphid assay may be any such assay known to the art, e.g., asdescribed in Hill, C. B., et al., Resistance to the soybean aphid insoybean germplasm, (2004) Crop Science 44:98-106, Hill, C. B., et al.,Resistance of Glycine species and various cultivated legumes to thesoybean aphid (Homoptera: Aphididae), (2004) J. Economic Entomology97:1071-1077, or Li, Y., et al., Effect of three resistant soybeangenotypes on the tecunalry, mortality, and maturation of soybean aphid(Homoptera: Aphididae), (2004) J. Economic Entomology 97:1106-1111, oras described in the Examples hereof.

One example of an antixenosis resistance assay includes placing aphidsor aphid-infested plant parts on VC or V1 stage plants and rating aphidpopulation and plant damage weekly. For example, in certain examples,numerous viviparous alate adult females are placed on newly expandedunifoliates with a moistened camel's hair paintbrush, the plants arearranged in a randomized design within a tray, and the aphid resistanceis evaluated at 7 and 14 days after infestation, using an antixenosisrating scale. One example of such an antixenosis scale is a 1-9 ratingscale wherein:

9=Equivalent or better when compared to a resistant check—No aphids onthe plant;

7=Very little damage, only a few aphids found on the plant;

5=Moderately Susceptible;

3=Major damage, including stunting and foliar stress; and

1=Plants are completely covered—Severe damage, including severe stuntingand necrosis; equivalent or worse when compared to a susceptible check.

One example of an antibiosis resistance assay includes placing onedouble-sided sticky cage containing two alate adult females on eachunifoliate of plants at the V1 stage and then placing a piece of organdycloth over the cage to restrict the aphids' movements. This is done forboth the plant variety to be tested and a plant variety known to besusceptible. The aphids are then allowed to reproduce for 96 hours and,at the end of this period, the cages are removed and counts performed onthe surviving and deceased aphids to determine the antibiosis resistanceof the plants tested. Plants with a high rate of nymphal production areclassified as susceptible. Plants with some nymphs, but withstatistically lower nymphal populations compared to the susceptiblecheck are classified as moderately resistant. Plants with no nymphproduction within the sticky cages and dead or unhealthy in appearanceadults are classified as resistant.

Automated Detection/Correlation Systems, Kits, and Nucleic Acids:

In some examples, a kit or an automated system for detecting markers,Rag haplotypes, and marker profiles, and/or correlating the markers, Raghaplotypes, and marker profiles with a desired phenotype (e.g.,resistance) are provided. Thus, a typical kit or system can include aset of marker probes or primers configured to detect at least onefavorable allele of one or more marker locus associated with resistanceor improved resistance to a soybean aphid infestation, for instance afavorable Rag haplotype or marker profile. These probes or primers canbe configured, for example, to detect the marker alleles noted in thetables and examples herein, e.g., using any available allele detectionformat, such as solid or liquid phase array based detection,microfluidic-based sample detection, etc. The systems and kits canfurther include packaging materials for packaging the probes, primers,or instructions, controls such as control amplification reactions thatinclude probes, primers or template nucleic acids for amplifications,molecular size markers, or the like.

A typical system can also include a detector that is configured todetect one or more signal outputs from the set of marker probes orprimers, or amplicon thereof, thereby identifying the presence orabsence of the allele. A wide variety of signal detection apparatus areavailable, including photo multiplier tubes, spectrophotometers, CCDarrays, scanning detectors, phototubes and photodiodes, microscopestations, galvo-scans, microfluidic nucleic acid amplification detectionappliances and the like. The precise configuration of the detector willdepend, in part, on the type of label used to detect the marker allele,as well as the instrumentation that is most conveniently obtained forthe user. Detectors that detect fluorescence, phosphorescence,radioactivity, pH, charge, absorbance, luminescence, temperature,magnetism or the like can be used. Typical detector examples includelight (e.g., fluorescence) detectors or radioactivity detectors. Forexample, detection of a light emission (e.g., a fluorescence emission)or other probe label is indicative of the presence or absence of amarker allele. Fluorescent detection is generally used for detection ofamplified nucleic acids (however, upstream and/or downstream operationscan also be performed on amplicons, which can involve other detectionmethods). In general, the detector detects one or more label (e.g.,light) emission from a probe label, which is indicative of the presenceor absence of a marker allele. The detector(s) optionally monitors oneor a plurality of signals from an amplification reaction. For example,the detector can monitor optical signals which correspond to “real time”amplification assay results.

System or kit instructions that describe how to use the system or kit orthat correlate the presence or absence of the favorable allele with thepredicted resistance are also provided. For example, the instructionscan include at least one look-up table that includes a correlationbetween the presence or absence of the favorable alleles, haplotypes, ormarker profiles and the predicted resistance or improved resistance. Theprecise form of the instructions can vary depending on the components ofthe system, e.g., they can be present as system software in one or moreintegrated unit of the system (e.g., a microprocessor, computer orcomputer readable medium), or can be present in one or more units (e.g.,computers or computer readable media) operably coupled to the detector.As noted, in one typical example, the system instructions include atleast one look-up table that includes a correlation between the presenceor absence of the favorable alleles and predicted resistance or improvedresistance. The instructions also typically include instructionsproviding a user interface with the system, e.g., to permit a user toview results of a sample analysis and to input parameters into thesystem.

Isolated nucleic acids comprising a nucleic acid sequence coding forresistance to soybean aphid, or sequences complementary thereto, arealso included. In certain examples, the isolated nucleic acids arecapable of hybridizing under stringent conditions to nucleic acids of asoybean cultivar resistant to soybean, for instance to particular SNPsthat comprise a Rag haplotype or marker profile. Vectors comprising suchnucleic acids, expression products of such vectors expressed in a hostcompatible therewith, antibodies to the expression product (bothpolyclonal and monoclonal), and antisense nucleic acids are alsoincluded.

As the parental line having soybean aphid resistance, any line known tothe art or disclosed herein may be used. Also included are soybeanplants produced by any of the foregoing methods. Seed of a soybeangermplasm produced by crossing a soybean variety having a Rag haplotypeor marker profile associated with soybean aphid resistance with asoybean variety lacking such Rag haplotype or marker profile, andprogeny thereof, is also included.

The present invention is illustrated by the following examples. Theforegoing and following description of the present invention and thevarious examples are not intended to be limiting of the invention butrather are illustrative thereof. Hence, it will be understood that theinvention is not limited to the specific details of these examples.

EXAMPLES Aphid Colonies:

The four biotype colonies of soybean aphids are maintained in a growthchamber at the Soybean insect Containment Facility (Dallas Center,Iowa). The colonies are maintained on a continuous supply of soybeanvariety 90M60. Two colonies of Urbana, Ill. (biotype 1) and Wooster,Ohio (biotype 2) were obtained from Brian Diers at the University ofIllinois. Biotype 3 (Indiana) was obtained from Curtis B. Hill at theUniversity of Illinois. Lime Springs, Iowa (biotype X) was collectedfrom soybean fields in Limes Springs, Iowa. The colonies are maintainedin isolated tents to avoid mixing.

Rag1-a/Rag2-b Stack

A population of 736 segregating plants was developed by crossing plantsthat carried (Rag-1a) from 95B97 and LD08-89051a (Rag2-b). The progenywere screened with the four biotypes to determine the efficacy of thestack compared to the single gene resistance. The 95B97 and LD08-89051adonors possess antibiosis resistance to some of the biotypes (Table 3).

Choice Bioassay (Antixenosis):

The choice tests were conducted in a growth chamber with temperaturesbetween 22 and 25° C. with a 16 hour photoperiod. The segregatingpopulation was planted in Cone-tainers™ (Stuewe and Sons, Inc., Tangent,Oreg.) and infested at the V1 growth stage. Seven viviparous apterousadult females were placed on the newly expanded unifoliates with amoistened camel's hair paintbrush. The plants were arranged incompletely randomized design within a tray including a susceptible checkand the two resistant donor lines. The trays were placed within a waterbath inside a Bioquip tent to isolate the aphids. The trays were wateredfrom the bottom to avoid disrupting the aphid feeding. The aphidresistance was evaluated at 7 and 14 days after infestation, using a 1-9antixenosis rating.

9=Equivalent or better when compared to the resistant check—No aphids onthe plant

7=Very little damage, only a few aphids found on the plant.

5=Moderately Susceptible

3=Major damage, including stunting and foliar stress

1=Plants are completely covered. Severe damage, including severestunting and necrosis; equivalent or worse when compared to thesusceptible

Non-Choice Bioassay (Antibiosis):

At the same time the plants were infested in the antixenosis bioassay,the antibiosis bioassay was conducted. To determine antibiosisresistance, a non-choice test was conducted (i.e., a test wherein theaphids have no choice but to either feed on the plant or starve todeath). The non-choice bioassay was conducted using the sameenvironmental conditions as described above in the choice bioassay. Atthe V1 stage, one double-sided sticky cage was placed on eachunifoliate. Using a moistened paintbrush, two viviparous apterous adultfemales were placed within the cage and a piece of organdy cloth wasplaced over the cage to restrict the aphids' movements. The aphids wereallowed to reproduce for 7 days and then the survival, death, andfecundity of the aphids within the cage were recorded. The fecundity wascalculated as the mean number of surviving nymphs produced within a cageduring the 96 hour period for each plant introduction. Plants that had ahigh rate of nymphal production were classified as susceptible. Plantswith some nymphs, but with statistically lower populations compared tothe susceptible check were classified as moderately resistant. Plantswith no nymph production within the sticky cages and dead or unhealthyin appearance adults were classified as resistant. The 736 were leafpunched to determine the genotypic class.

TABLE 3 Aphid resistance phenotype and genotype data for selected PIsCountry Antibiosis Results Antixenosis Results Variety MG of Origin 1 23 X Y 1 2 3 X Y Rag Haplotype 95B97 Pioneer R S M R S R S M R S Rag1-aDowling VIII US R S M R S R S M R S Rag1-a LD08-89068a III Illinois R RS S S R R S S S Rag2-b PI200538 VIII Japan R R S S S R R S S S Rag2-bRag1/Rag2 US R R R R S R R R R S Rag1-a & Rag2-b stack PI567666 IV ChinaR R R R R R R R R R Rag1-b & Rag3-b PI567622 IV China R R R R R R R R RR Rag1-b & Rag3-b PI219652 VII Indonesia S S S S S R R R R R Rag1-c &Rag3-d PI219655 VII Indonesia S S S S S R R R R R Rag1-c & Rag3-d

These results demonstrate that stacking Rag haplotypes results in anunexpected synergistic effect. For instance, while neither the Rag1-anor the Rag2-b donors exhibited a resistant antibiosis or antixenosisphenotype in regards to biotype 3, the Rag1-a/Rag2-b stack plants didexhibit a resistant phenotype to biotype 3 in both the antibiosis andantixenosis assays.

What is claimed is:
 1. A method of improving resistance to one or moresoybean aphid biotypes in a soybean plant, line, or strain, the improvedresistance comprising one or more of improved antibiosis resistance andimproved antixenosis resistance; the method comprising: a) selecting afirst soybean plant or germplasm, or a progeny thereof, wherein saidfirst soybean plant, germplasm, or progeny comprises a favorable alleleof at least one of Rag1, Rag2, and Rag3; b) selecting a second soybeanplant or germplasm, or a progeny thereof, wherein said second soybeanplant, germplasm, or progeny comprises a favorable allele of at leastone of Rag1, Rag2, and Rag3 different than that of said first soybeanplant, germplasm, or progeny; and c) crossing said first soybean plant,germplasm, or progeny with said second soybean plant, germplasm, orprogeny.
 2. The method of claim 1, wherein said first soybean plant,germplasm, or progeny and said second soybean plant, germplasm, orprogeny collectively comprise a favorable allele of all three of Rag1,Rag2, and Rag3.
 3. The method of claim 1, wherein the improved soybeanaphid resistance comprises both improved antibiosis resistance andimproved antixenosis resistance.
 4. The method of claim 1, wherein theimproved soybean aphid resistance comprises improved resistance to atleast two soybean aphid biotypes.
 5. The method of claim 1, wherein theimproved soybean aphid resistance comprises improved resistance to atleast three of soybean aphid biotypes 1, 2, 3, X, and Y.
 6. The methodof claim 1, wherein the selection further comprises detecting saidfavorable allele, said detecting step comprising amplifying a markernucleic acid or a portion of the marker nucleic acid and detecting theresulting amplified marker nucleic acid.
 7. The method of claim 6,wherein the amplifying comprises performing a polymerase chain reaction(PCR) using one or more nucleic acid from the plant as a template in thePCR.
 8. The method of claim 1, wherein said favorable alleles areselected from the group consisting of Rag1-b, Rag1-c, Rag2-d, Rag3-b,and Rag3-d.
 9. The method of claim 1, wherein said favorable allele insaid first soybean plant comprises Rag1-a, and wherein said favorableallele in said second soybean plant comprises Rag2-b.
 10. The method ofclaim 6, wherein said amplifying comprises amplification of one or moreportions of the soybean genome selected from the group consisting of SEQID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 84,89, 94, 103, 114, 121, 126, 131, and
 136. 11. The method of claim 10,wherein said amplification comprises providing one or more nucleic acidprimers, wherein said primers comprise a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21,22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51, 52, 56, 57, 61, 62, 66,67, 71, 72, 76, 77, 80, 81, 85, 86, 90, 91, 95, 96, 99, 100, 104, 105,108, 109, 110, 111, 115, 116, 119, 120, 122, 123, 127, 128, 132, and133.
 12. The method of claim 1, wherein said detecting compriseshybridization with one or more nucleic acid probes, wherein said probescomprise a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 3, 4, 8, 9, 13, 14, 18, 19, 23, 24, 28, 29, 33, 34, 38, 39,43, 44, 48, 49, 53, 54, 58, 59, 63, 64, 68, 69, 73, 74, 78, 79, 82, 83,87, 88, 92, 93, 97, 98, 101, 102, 106, 107, 112, 113, 117, 118, 124,125, 129, 130, 134, 135, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 84, 89, 94, 103, 114, 121, 126, 131, and
 136. 13. A plantproduced by the method of claim
 1. 14. A method of selecting a soybeanplant or germplasm that displays improved resistance to one or moresoybean aphid biotypes, the improved resistance comprising one or moreof improved antibiosis resistance and improved antixenosis resistance,the method comprising: detecting in a first soybean plant or germplasm,or a part thereof, a favorable allele of at least two of Rag1, Rag2, andRag3; and selecting the first soybean plant or germplasm, or selecting aprogeny of the first soybean plant or germplasm.
 15. The method of claim14, wherein the detecting involves detection of a favorable allele ofall three of Rag1, Rag2, and Rag3.
 16. The method of claim 14, whereinthe improved soybean aphid resistance comprises both improved antibiosisresistance and improved antixenosis resistance.
 17. The method of claim14, wherein the improved soybean aphid resistance comprises improvedresistance to at least two soybean aphid biotypes.
 18. The method ofclaim 14, wherein the improved soybean aphid resistance comprisesimproved resistance to at least three of soybean aphid biotypes 1, 2, 3,X, and Y.
 19. The method of claim 14, wherein said favorable alleles areselected from the group consisting of Rag1-b, Rag1-c, Rag2-d, Rag3-b,and Rag3-d.
 20. The method of claim 14, wherein said favorable allelescomprise Rag1-a and Rag2-b.
 21. The method of claim 14, wherein thedetecting comprises amplifying a marker nucleic acid or a portion of themarker nucleic acid and detecting the resulting amplified marker nucleicacid.
 22. The method of claim 21, wherein the amplifying comprisesperforming a polymerase chain reaction (PCR) using one or more nucleicacid from the plant as a template in the PCR.
 23. The method of claim22, wherein said amplifying comprises amplification of one or moreportions of the soybean genome selected from the group consisting of SEQID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 84,89, 94, 103, 114, 121, 126, 131, and
 136. 24. The method of claim 13,wherein said amplification comprises providing one or more nucleic acidprimers, wherein said primers comprise a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21,22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51, 52, 56, 57, 61, 62, 66,67, 71, 72, 76, 77, 80, 81, 85, 86, 90, 91, 95, 96, 99, 100, 104, 105,108, 109, 110, 111, 115, 116, 119, 120, 122, 123, 127, 128, 132, and133.
 25. The method of claim 14, wherein said detecting compriseshybridization with one or more nucleic acid probes, wherein said probescomprise a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 3, 4, 8, 9, 13, 14, 18, 19, 23, 24, 28, 29, 33, 34, 38, 39,43, 44, 48, 49, 53, 54, 58, 59, 63, 64, 68, 69, 73, 74, 78, 79, 82, 83,87, 88, 92, 93, 97, 98, 101, 102, 106, 107, 112, 113, 117, 118, 124,125, 129, 130, 134, 135, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 84, 89, 94, 103, 114, 121, 126, 131, and
 136. 26. The methodof claim 14, further comprising crossing the selected first soybeanplant or germplasm with a second soybean plant or germplasm.
 27. Themethod of claim 21, wherein the second soybean plant or germplasmcomprises an exotic soybean strain or an elite soybean strain.
 28. Aplant selected by the method of claim
 14. 29. A plant produced by themethod of claim
 21. 30. A kit for selecting a soybean plant or germplasmthat displays improved resistance to one or more soybean aphid biotypes;the kit comprising: a) primers or probes for detecting a favorableallele of at least two of Rag1, Rag2, and Rag3; and b) instructions forusing the primers or probes for detecting the one or more favorablealleles and correlating the detected alleles with predicted improvedresistance to aphids.
 31. The kit of claim 30, wherein the kit comprisesprimers or probes for detecting a favorable allele of all three of Rag1,Rag2, and Rag3.
 32. The plant of any one of claims 13, 28, 29, whereinsaid plant comprises stacked Rag haplotypes selected from the groupconsisting of a) Rag1-a/Rag2-b; b) Rag1-b/Rag3-b; and c) Rag1-c/Rag3-d.33. An isolated polynucleotide capable of detecting: a) a marker locusselected from the group consisting of S15354-001-Q001, S14181-1-Q1,S13871-1-Q1, S14161-1-Q10, S09515-1-Q1, S14151-1-Q1, S14151-2-Q4,S07164-1-Q12, S14182-1-Q1, S00812-1-A, S02780-1-A, S01190-1-A,S14761-001-Q001, S14771-001-Q001, S07165-1-Q3, S14778-001-Q001,S01164-1-Q1 S13662-1-Q3/Q6, S13663-1-Q1, S11411-1-Q1, S13664-1-Q1/Q002,S13672-1-Q1/Q2/Q3, S13674-1-Q1/Q007, S13675-2-Q1; or b) a SNP locilocated at a physical position selected from the group consisting of5453161, 5516385, 5516818, 5598980, 5602544, 5605203, 5605275, 5608106,5630404, 6754454, and 6671535 on LG-M of the soybean genome, 28187733,28829625, 28837383, 29097652, 29678319, and 29825175 on LG-F of thesoybean genome; and 5140274, 5919650, 5960726, 6066531, 6231641,6524877, and 6542422 on LG-J of the soybean genome.
 34. The isolatedpolynucleotide of claim 33, wherein the polynucleotide comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:1-136.
 35. A kit for detecting or selecting at least one soybean plantwith stacked Rag haplotypes, the kit comprising: a) primers or probesfor detecting two or more marker loci associated with one or morequantitative trait loci associated with improved aphid resistance,wherein the primers or probes comprise an isolated nucleic acid of claim24; and b) instructions for using the primers or probes for detectingthe two or more marker loci and correlating the detected marker lociwith predicted improved resistance to aphid infestation.